Method for removing glucosinolates from oilseed meals

ABSTRACT

A process to remove the glucosinolates of oilseed meals, such as  Brassica carinata  oilseed meals, is provided. In one embodiment, exogenous myrosinase is used to convert the glucosinolates to volatile isothiocyanate compounds, which can then be removed under conditions of mild heat and negative pressure. In another embodiment, heat and pressure are used to remove glucosinolates from  Brassica carinata  oilseed. The processed meals may contain less than 80% of their starting levels of glucosinolates and may be suitable for use in various applications, including as animal feeds.

FIELD

The present application relates to methods of removing glucosinolatesfrom oilseed meals.

BACKGROUND

Brassica carinata is a member of the Brassicaceae (formerly Cruciferae)family, commonly known as the mustard family The genus Brassica is amember of the tribe Brassiceae in the mustard family (Warwick et al.2009). In addition to B. carinata, the Brassica genus includes severaleconomically important oilseed crop species: B. juncea (L). Czern.(brown mustard), B. napus L. (rape, Argentine canola), B. nigra (L.) W.D. J. Koch (black mustard), and B. rapa L. (field mustard, Polishcanola). The genus Brassica also includes B. oleracea L. food crops,including cabbage, broccoli, cauliflower, Brussels sprouts, kohlrabi andkale. The six Brassica species are closely related genetically, asdescribed in the Triangle of U (Nagaharu, 1935, reviewed in Branca andCartea, 2011). Brassica carinata is an amphidiploid (BBCC, 2n=34)thought to be derived from interspecific hybridization of the diploidspecies B. nigra L. (BB, 2n=16) and B. oleracea L. (CC, 2n=18; Prakashet al., 2012).

Recent breeding efforts have focused on the development of new oilseedfeedstock crop for biofuels (e.g. ethanol, biodiesel, bio-jet fuel),bio-industrial uses (e.g., bio-plastics, lubricants) and specialty fattyacids (e.g., erucic acid); Taylor et al., 2010. Chief among these aremembers of the Brassicaceae family: Brassica carinata (Ethiopianmustard), Camelina sativa (False flax), Thlaspi arvense (pennycress) andCrambe abyssinica (Crambe). These species have been selected not onlybecause of their potential for providing a high quality feedstock oilbut also because of their ability to be grown sustainably in manyregions of North America and elsewhere with minimal need for land usechange nor displacement of other crops grown for food (Drenth et al.,2014, 2015).

Brassica carinata was assessed in the mid-1980s as a potentialalternative oilseed crop for North America (Getinet, 1986; Getinet etal., 1996). In Spain and Italy, Brassica carinata seed oil is used forbiofuel (Bouaid et al., 2005; Cardone et al., 2002, 2003; Gasol et al.,2007, 2009) and as a bio-industrial feedstock with many uses (e.g. inlubricants, paints, cosmetics, plastics). In Canada and the US, Brassicacarinata is also being exploited as a biofuel feedstock (Blackshaw etal., 2011; Taylor et al., 2010; Marillia et al., 2014; Drenth et al.,2014, 2015), and oil extracted from Brassica carinata seed has been usedfor the production of green bio-diesel and bio-jet fuel. In October2012, experimental aviation flights by the National Research Council ofCanada using the world's first 100% bio-jet fuel were successful(National Research Council of Canada 2013).

While the oils produced by Brassica carinata and other novel oilseedcrops are of great value largely because of their utility as anindustrial feedstock, the meal produced as part of the oil extractionprocess is a potentially valuable co-product in its own right. Forexample, the protein rich and low fiber meal can be used as an additivein livestock and poultry feed rations. Its only limitation as a feedadditive is its relatively high content of anti-nutritional compounds,chiefly glucosinolate.

Glucosinolates constitute a large family of over 100 related moleculeswith a common sulfur containing core structure and with side chains ofvarying size and chemistry (Fahey et al., 2001; Halkier and Gershenzon,2006). While glucosinolates are found in many plant structures (leaf,vascular tissue, stem, root, and flowers, to cite some examples), theyare accumulated in high concentrations in the seed (Bellostas et al.,2004). This is particularly true for the oil seed brassicas. Thesecompounds and their metabolites can impact the taste of the meal,reducing its palatability and in some cases (dependant on the type ofglucosinolate and glucosinolate metabolites present) can also adverselyimpact the animal's health directly. For example, hydrolysis products ofbeta hydroxyalkenyl glucosinolates have been shown to possessgoitrogenic activity in animal models (reviewed in Fahey et al., 2001).This is particularly an issue in monogastic animals such as swine, butpoultry and cattle can be susceptible to varying degrees. Thusglucosinolate reduction in oil seed meal is an important and desirableobjective and can have significant benefits in terms of meal value.

Current commercial varieties of Brassica carinata seed containappreciable levels of glucosinolate (60-100 mmol/g). The predominantglucosinolate species in Brassica carinata seed is sinigrin (2-propenylglucosinolate, also known as allyl glucosinolate) comprisingmore than 90% of the total glucosinolate content. This is quite distinctfrom other commercial brassica oilseeds such as canola type Brassicanapus, which although lower in overall seed glucosinolate levels (5-12μmol/g) than Brassica carinata, has a very different chemical profile,with progoiterin (2-(R)-2-Hydroxy-3-butenylglucosinolate), gluconapin(3-Butenylglucosinolate) and 4 hydroxyglucobrassicin (4 hydroxyindolemethylglucosinolate) being the predominant species and with little or nosinigrin (Xin et al., 2014). Due to the relatively high levels ofglucosinolate in the seed of Brassica carinata, carinata meal usage as afeed additive is currently limited to cattle and other ruminant speciesthat are relatively tolerant of glucosinolate. Even in this casehowever, the amounts of carinata meal that can be included are limitedto 10 percent due to the glucosinolate levels. The amounts of camelinameal that can be included in beef cattle feed rations are similarlylimited.

Several approaches have been taken to achieve reduction in glucosinolatelevels in oilseeds. One group of approaches involves processing methodsto physically or chemically remove glucosinolates during the processingof the oilseed into its end products while the other involvesmanipulation of the oilseed varieties through breeding and selection toproduce varieties that accumulate much lower levels of glucosinolatewithin the seed itself. The latter approach has largely supplanted thefirst since, over a number of years, low glucosinolate varieties havebeen successfully obtained in many brassica oilseed species, includingB. napus, B. rapa and B. juncea. Thus, the requirement for reduction ofglucosinolate levels by processing has largely been rendered superfluousfor specific varieties within these species, but for many otherBrassicaceae species, processing to reduce glucosinolate levels remainsa viable option. To date there has been no description on how suchprocesses can be used to reduce glucosinolate content of Brassicacarinata meal. In particular, the art does not describe the processesand temperatures that could be applied to Brassica carinata seed that issubject to oil extraction using solvent to produce a low glucosinolatemeal product. It is an object of the present invention to provide anovel method for obtaining Brassica carinata solvent extracted meal withreduced glucosinolates.

Processing methodologies to reduce glucosinolates can be divided intotwo general classes, those that focus on the direct removal ofglucosinolate and those that rely on conversion of the glucosinolate toa metabolic byproduct, isothiocyanate, and then subsequent physicalremoval of the isothiocyanate. Before considering these two broadclasses of removal processes in greater detail. It would be instructiveto review the current state of the art in oilseed processing atindustrial scale.

Processing of brassica oilseeds to extract the oil involves multiplesteps. Typically the seeds are cleaned then crushed in a roller mill togenerate flakes of 0.3-0.38 mm in thickness. The flaked seed thenundergoes a process known as cooking whereby it is conveyed to a heateddrum where the flakes are cooked at elevated temperatures (typicallyfrom 70-90° C.) for up to 20 min. The cooking helps to reduce theviscosity of the oil to allow for more efficient extraction insubsequent steps, but it also inactivates the endogenous myrosinaseenzyme. Cooked seed flakes are then pressed in a series of screw pressesor expellers which can remove 50-60% of the oil. Aside from the oilwhich is removed for further processing, the pressing produces a mealcake that is ideal for solvent extraction. Using several cycles ofcountercurrent extraction, the meal cake is treated with hexane toremove the residual oil from the meal. The meal is then transferred to adesolventizer-toaster where it is heated to remove remaining hexane; thefinal step of the process, called toasting, involves injection of steaminto the meal to remove the last traces of hexane. The meal is thencooled and dried by blowing forced air through it.

In some cases, the seed can also be processed using a cold pressmethodology which is similar to above except it does not involve the useof hexane to remove residual oil from the oil cake, resulting in a mealwith much higher oil composition.

When canola seed is crushed to yield oil and meal, seed glucosinolatereduction is not a preeminent consideration in the design of theprocess. All steps involving heating, i.e. the cooking step as well asthe desolventizing, toasting step, are designed to require the lowestheat required to achieve their respective ends. This is to achieve thebest balance of oil yield and meal quality, the latter being mostsensitive to the deleterious effect of high heat on protein levels andquality. When the seed to be processed is not canola quality, however,consideration must be given to means of reducing the endogenousglucosinolates to allow for improved meal and oil quality. Asglucosinolates are to some extent heat labile, the cooking step can beemployed for glucosinolate removal. Increasing the cooking temperatureto as high as 120° C. has been employed to reduce glucosinolate levels;however, this can have deleterious effects on heat labile proteins ofthe meal, reducing its value substantially. Other modifications of thecrushing process have been described with the goal of reducingglucosinolate in meal. The use of an extruder apparatus, used instead ofthe typical screw process, has been shown to be beneficial in reductionof glucosinolates from rapeseed meal. However, these processes have notbeen previously described for Brassica carinata.

There are many examples in the literature of processes to reduceglucosinolate from meal that has already been processed for removal ofoil, i.e. that had previously gone through a process similar to thatdescribed in the previous paragraphs. The earliest attempts at directreduction of glucosinolates involved application of heat or heatcombined with methods to reduce the particle size of the meal (i.e.micronization and extrusion technologies). Heating in the form ofmicrowave exposure was shown to reduce glucosinolate levels viadegradation (Aumaitre et al., 1989). The authors estimated thatmicrowave heating methodologies can result in up to 25% reduction inglucosinolate levels. Application of heating, micronization andtreatment in an extruder were each shown to be useful for glucosinolatereduction and the magnitude of the reduction was increased if chemicalagents such as alkali or ammonium were added to the meal (Fenwick etal., 1986). However, the authors of this study noted that the magnitudeof glucosinolate reduction was greatest under conditions which alsoaffected the integrity of other nutritional components. For example,application of excessive heat (however applied) while significantlyreducing glucosinolate levels, also hastened degradation of proteins viathe Maillard reaction (Anderson-Haferman et al., 1993). Such unwantedeffects on protein quality are a particular property of this class ofglucosinolate reduction strategies. It would also in many casesnecessitate investment in new equipment and additional processing steps,which would affect the cost of processing and ultimately of the mealitself. As above, the art is void of such approaches with Brassicacarinata.

Processes have been developed to remove glucosinolates from meal basedon their interactions with aqueous solvents (reviewed by Tripathi andMishra, 2007). Significant loss of glucosinolates due to hydrolysis canoccur during prolonged soaking in water. Supplementation of soakingbuffer with metal ions (such as Cu++) could further potentiate theremoval of glucosinolates. While the process is economical, losses ofdry matter during the soaking can affect the quality of and quantity ofmeal for feed applications.

It was recognized quite early that under the right conditionsglucosinolate content of the meal could be converted to isothiocyanatealmost quantitatively by action of myrosinase, which could subsequentlybe removed by a variety of methods. In fact the action of myrosinase onglucosinolate is tightly controlled during the seed crushing process.Normally sequestered within the cellular structure, myrosinase ismobilized by processes which physically disrupt the seed's structure andintegrity, such as crushing. This mobilization brings it into contactwith the glucosinolate of the meal fraction and under the appropriateconditions of temperature, pH and humidity could quantitatively convertthe glucosinolate to isothiocyanate. While this would be beneficial asregards meal quality it would have other less desirable consequences.For example, the isothiocyanate, being very lipid soluble couldpotentially adulterate the oil component, and result in an unacceptablyhigh oil sulphur content.

In addition, myrosinase catalyzes the conversion of sinigrin (allylglucosinolate) to allyl isothiocyanate. Allyl isothiocyanate is volatile(Dai and Lim, 2014) and is also known as volatile oil of mustard. Allylisothiocyanate is highly pungent, and is responsible for the pungenttaste of horse radish and wasabi root. In pure form it can be toxic,acting as an irritant to skin and mucous membranes. Isothiocyanates alsoimpart a pungent taste to a feed ration, which reduces its palatabilityand adversely affects the livestock's intake of the meal.

For these reasons, the oilseed crushing process described earlierincorporates the heating step before crushing in part to inactivate themyrosinase, ensuring that the conversion of glucosinolates toisothiocyanates does not take place.

Nevertheless, others have described processes whereby myrosinase couldbe used to advantage to convert glucosinolate to isothiocyanate at laterstages of the crushing process (i.e. after the removal of the oil hasbeen completed). In this scenario, an exogenous source of myrosinase isadded back to the processed meal and allowed to react with theendogenous glucosinolate under optimized conditions. The releasedisothiocyanate could be then extracted from the meal using a variety ofdifferent solvents (see U.S. Pat. No. 4,244,973). Much like the directglucosinolate removal processes, the utility of this approach is greatlyinfluenced by the additional costs and equipment required to process themeal and the potential deleterious effects of the solvent treatment onmeal quality.

With a new generation of oilseed crops being developed to provide oilbased feedstock for industrial purposes, the economic value of thesecrops can be greatly enhanced if other value added by-products of theoil extraction process can be commercially exploited. Brassica carinata,for example, produces a seed oil that is highly valued as an industrialfeedstock while its meal rivals soybean meal in terms of protein qualityand low fiber content. If the levels of glucosinolate in carinata mealcould be reduced to those of double zero canola quality meal, it wouldsignificantly increase the market value for carinata meal as a feedadditive. While efforts to develop low glucosinolate varieties ofcarinata are ongoing, there exists a need to develop an economical andeffective process to reduce the glucosinolate levels of existing sourcesof carinata meal. The process should be easily adaptable to existing oilcrushing plants in terms of cost, time and equipment so as not toconstitute a deterrent to its adoption by the industry. Moreover, theprocess should be sufficiently gentle so as not to compromise theadvantage that carinata holds in terms of protein content over otheroilseed meals. Such a process could also be adapted to other oilseeds byvirtue of their common characteristics. In this regard, it isinstructive to note that even the best current varieties of canola havesmall but measurable quantities of glucosinolates remaining in theirmeal and that the ability to remove these in a cost effective way mayenable new products and new markets for canola meal as well.

While the current art teaches how glucosinolate levels can be reduced inoilseed meals, the methods of reduction invariably involve processesthat that can adversely affect the integrity of the meal proteinconstituents either through denaturation or via extractive losses. Asprotein constitutes the most important nutritional component of meal,processes that affect the protein content or quality can also affect thevalue of the meal.

SUMMARY

In one embodiment, the present invention provides a process for removingat least one glucosinolate from a meal fraction of oilseed comprising:(a) treating the meal fraction of oilseed with exogenous myrosinase toconvert the at least one glucosinolate to a volatile isothiocyanate; and(b) removing the volatile isothiocyanate from the treated meal fractionof oilseed under conditions of mild heat and negative pressure.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of oilseed, wherein step (a) and step (b) occur simultaneouslyor sequentially.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of oilseed, wherein the time for step (a) and step (b) isgreater than 2 min, 5 min, 10 min, 15 min, 20 min, 30 min, 1 h, 2 h, 3h, or 4 h, and less than 9 h, 10 h, 11 h, or 12 h.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of oilseed, wherein the process further comprises regulatingthe temperature during step (a) and step (b) to prevent the temperaturefrom exceeding 45° C., 50° C., 55° C., 60° C., 65° C., or 70° C.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of oilseed, wherein the process further comprises carrying outstep (a) and step (b) in a reaction vessel under negative pressure ofover 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%vacuum and evacuating volatile substances from the reaction vessel.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of oilseed, wherein the process further comprises increasingthe temperature to over 60° C., 65° C., 70° C., 75° C., 80° C., 85° C.,or 90° C., but to less than 95° C.,100° C., 105° C., 110° C., or 115°C., following step (a) and step (b) and further incubating the mealfraction of oilseed until the moisture content of the meal fraction ofoilseed decreases to less than 20%, 18%, 16%, 14%, 12%, or 10%.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of oilseed, wherein the process further comprises increasingthe temperature to over 60° C., 65° C., 70° C., 75° C., 80° C., 85° C.,or 90° C., but to less than 95° C,100° C., 105° C., 110° C., or 115° C.,following step (a) and step (b) and further incubating the meal fractionof oilseed until the moisture content of the meal fraction of oilseeddecreases to less than 20%, 18%, 16%, 14%, 12%, or 10%, and wherein theprocess further comprises carrying out the further incubation in areaction vessel under negative pressure of over 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 98%, or 99% vacuum and evacuating volatilesubstances from the reaction vessel.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of oilseed, wherein the process further comprises continuouslymixing the meal fraction of oilseed and exogenous myrosinase.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of oilseed, wherein the process further comprises preheatingthe meal fraction of oilseed to between 25° C. to 40° C. prior to step(a).

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of oilseed, wherein the process further comprises providing theexogenous myrosinase in the form of a triggering solution comprising aslurry of defatted meal in water.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of oilseed, wherein the process further comprises providing theexogenous myrosinase in the form of a triggering solution comprising aslurry of defatted meal in water, and wherein the defatted meal is fromoilseed of a plant species of the Brassicaceae family

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of oilseed, wherein the process further comprises providing theexogenous myrosinase in the form of a triggering solution comprising aslurry of defatted meal in water, and wherein the defatted meal is fromoilseed of Sinapis alba or Brassica carinata.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of oilseed, wherein the process further comprises providing theexogenous myrosinase in the form of a triggering solution comprising aslurry of defatted meal in water, and wherein the triggering solutionfurther comprises ascorbic acid.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of oilseed, wherein the process further comprises addingtriggering solution to the meal fraction of oilseed in a ratio of 0.5:1,0.55:1, 0.6:1, 0.65:1, or 0.7:1 (w/w) triggering solution: meal fractionof oilseed.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of oilseed, wherein the meal fraction of oilseed results fromhexane extraction of oilseeds.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of oilseed, wherein the meal fraction of oilseed results fromhexane extraction of oilseeds, wherein the hexane extraction was carriedout on flaked and cooked oilseed, and wherein the cooking was carriedout at a temperature greater than 70° C., 75° C., 80° C., 85° C., 90°C., 95° C., 100° C., 105° C., 110° C., 115° C., 120° C., 125° C., 130°C., 135° C., or 140° C., but less than 150° C., 155° C., 160° C., 165°C., 170° C., 175° C., or 180° C.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of oilseed, wherein the meal fraction of oilseed results fromhexane extraction of oilseeds, wherein the hexane extraction was carriedout on flaked and cooked oilseed, and wherein the duration of cookingwas at least 10, 12, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28 or 30minutes, but less than 60, 70, 80, 90, or 100 minutes.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of oilseed, wherein the meal fraction of oilseed results fromhexane extraction of oilseeds, and wherein the hexane extraction wascarried out on flaked and cooked oilseed that had been pressed using anexpeller or a screw press.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of oilseed, wherein the meal fraction of oilseed results fromhexane extraction of oilseeds, wherein the hexane extraction was carriedout on flaked and cooked oilseed, and wherein the resultant meal cakeunderwent treatment in a desolventizer-toaster at a temperature of atleast 50° C., 55° C., 60° C., 65° C., 70° C., 75° C., 80° C., 85° C.,90° C., 95° C., 100° C., 105° C., 110° C., 115° C., 120° C., 125° C., or130° C., but less than 135° C., 140° C., 145° C., or 150° C.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of oilseed, wherein the reaction vessel is adesolventizer-toaster, and wherein step (a), step (b), and the furtherincubation are part of a desolventizer-toaster step of hexane extractionof oilseeds.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of oilseed, wherein the meal fraction of oilseed results fromcold press processing of oilseeds.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of oilseed, wherein the meal fraction of oilseed to be treatedis pelleted and homogenized by hammer-milling to a size of less than5.66 mm, 4.75 mm, 4.00 mm, 3.36 mm, 2.83 mm or 2.38 mm.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of oilseed, wherein the meal fraction of oilseed is fromoilseed of a plant species of the family Brassicaceae.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of oilseed, wherein the meal fraction of oilseed is fromoilseed of Brassica juncea, Brassica napus, Brassica rapa, Brassicacarinata, Brassica nigra, Cammelina sativa, Crambe abyssinica, orThlaspi arvense.

In another embodiment, the present invention provides a process forremoving at least one glucosinolate from a meal fraction of a Brassicacarinata oilseed comprising heating and applying pressure to the oilseedbefore, during, or after the extraction of oil.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of a Brassica carinata oilseed comprising heating and applyingpressure to the oilseed before, during, or after the extraction of oil,wherein the temperature during heating is greater than 70° C., 75° C.,80° C., 85° C., 90° C., 95° C., 100° C., 105° C., 110° C., 115° C., 120°C., 125° C., 130° C., 135° C., or 140° C., but less than 150° C., 155°C., 160° C., 165° C., 170° C., 175° C., or 180° C.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of a Brassica carinata oilseed comprising heating and applyingpressure to the oilseed before, during, or after the extraction of oil,wherein the duration of heating is at least 10, 12, 14, 15, 16, 17, 18,19, 20, 22, 24, 26, 28, or 30 minutes, but less than 60, 70, 80, 90, or100 minutes.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of a Brassica carinata oilseed comprising heating and applyingpressure to the oilseed before, during, or after the extraction of oil,wherein the process further comprises applying the pressure using anexpeller or a screw press.

In another embodiment, the present invention provides a process forremoving at least one glucosinolate from a meal fraction of a Brassicacarinata oilseed comprising: (a) heating and applying pressure to theoilseed before, during, or after the extraction of oil; (b) treating themeal fraction of oilseed with exogenous myrosinase to convert the atleast one glucosinolate to a volatile isothiocyanate; and (c) removingthe volatile isothiocyanate from the treated meal fraction of oilseedunder conditions of mild heat and negative pressure.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of a Brassica carinata oilseed comprising: (a) heating andapplying pressure to the oilseed before, during, or after the extractionof oil; (b) treating the meal fraction of oilseed with exogenousmyrosinase to convert the at least one glucosinolate to a volatileisothiocyanate; and (c) removing the volatile isothiocyanate from thetreated meal fraction of oilseed under conditions of mild heat andnegative pressure; wherein the temperature of heating in step (a) isgreater than 70° C., 75° C., 80° C., 85° C., 90° C., 95° C., 100° C.,105° C., 110° C., 115° C., 120° C., 125° C., 130° C., 135° C., or 140°C., but less than 150° C., 155° C., 160° C., 165° C., 170° C., 175° C.,or 180° C.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of a Brassica carinata oilseed comprising: (a) heating andapplying pressure to the oilseed before, during, or after the extractionof oil; (b) treating the meal fraction of oilseed with exogenousmyrosinase to convert the at least one glucosinolate to a volatileisothiocyanate; and (c) removing the volatile isothiocyanate from thetreated meal fraction of oilseed under conditions of mild heat andnegative pressure; wherein the duration of heating in step (a) is atleast 10, 12, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, or 30 minutes,but less than 60, 70, 80, 90, or 100 minutes.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of a Brassica carinata oilseed comprising: (a) heating andapplying pressure to the oilseed before, during, or after the extractionof oil; (b) treating the meal fraction of oilseed with exogenousmyrosinase to convert the at least one glucosinolate to a volatileisothiocyanate; and (c) removing the volatile isothiocyanate from thetreated meal fraction of oilseed under conditions of mild heat andnegative pressure; wherein the process further comprises applying thepressure in step (a) using an expeller or a screw press.

BRIEF DESCRIPTION OF THE FIGURES

In the figures, which illustrate embodiments of the invention by way ofexample only:

FIG. 1 is diagram of the enzymatic reaction converting glucosinolate toisothiocyanate catalyzed by myrosinase.

FIG. 2 is a schematic diagram of an illustrative apparatus that may beused for the batchwise removal of glucosinolate from oilseed meal.

DETAILED DESCRIPTION

With a new generation of oilseed crops, such as Brassica carinata, beingdeveloped to provide oil based feedstock for industrial purposes, thereexists a need to develop an economical and effective process to reducethe glucosinolate levels of the meal in order to be able to commerciallyexploit the meal, including, for example, as a feed additive for animalfeeds.

The process should be easily adaptable to existing oil crushing plantsin terms of cost, time and equipment so as not to constitute a deterrentto its adoption by the industry. Moreover the process should besufficiently gentle so as not to compromise the advantage that carinataholds in terms of protein content over other oilseed meals.

“Animal feeds” are those processed formulations that are fed tolivestock, as opposed to those sources of nutrition that animals mayforage for themselves. Animal feeds may be formulated to provide optimumnutrition for particular applications, such as maximizing weight gainand meat quality of beef cattle in the feed lot, or maximizing milkproduction of lactating dairy cattle. For poultry, feeds may beformulated to provide a consistent nutritional source to supplement oreven replace the variable nutritional quality obtained through foraging.While the composition of feeds may vary greatly based on animal,application and geography, a constant requirement is the addition ofsupplements to the base feed to improve its protein content. Typical ofsuch supplements are dried distiller grains, consisting of the spentfermented mash obtained from breweries or spirit distilleries, as wellas meal from soybean, canola meal, carinata or mustard, all obtainedafter seed crush and extraction of the oil fraction.

“Anti-nutritional” is a general description of a number of compoundsfound in Brassica and other seed meals that reduce the nutritionalbenefit of animal feed products in which the meal is used as anadditive. Glucosinolates and isothiocyanates are classified asanti-nutritionals since, when present in high enough concentrations,they impart a bitter and pungent taste to the feed ration, reducing itspalatability and adversely affecting the livestock's intake of the meal.

Glucosinolates

“Glucosinolates” are β-thioglucoside N-hydroxysulfates with a variableside chain (R) and a sulfur-linked β-d-glucopyranose moiety. Theyrepresent a large and heterogeneous family of naturally occurringcompounds; more than 120 varieties are known to occur in nature (Fahey,et al., 2001). Glucosinolates have the following chemical structure:

“Sinigrin” is the common name of allyl glucosinolate (or 2-propenylglucosinolate), where the R position has been substituted with an allylgroup. Sinigrin is the predominant glucosinolate species found inBrassica carinata and Brassica nigra seeds, and is also found, in lesseramounts in seeds of other Brassicaceae species. Sinigrin has thefollowing chemical structure:

Glucosinolates are found in many species of plants, particularly thosewithin the order Brassicales, but also among plants of the genusDrypetes and the genus Putranjiva (both genera of the Putranjivaceaefamily) They are accumulated to high levels in the seed, as well asother plant tissues. This is particularly true for the oil seedbrassicas. The glucosinolates found in meal samples of oilseeds includesinigrin, sinalbin, gluconapin, and gluconasturtin, among others, andtheir relative proportions can vary significantly depending on thespecies. These compounds and their metabolites can impact the taste ofthe meal, reducing its palatability and in some cases (dependant on thetype of glucosinolate and glucosinolate metabolites present) can alsoadversely impact the health of an animal that has consumed plantmaterial containing glucosinolates. Glucosinolate reduction in oil seedmeal is an important and desirable objective and can have significantbenefits in terms of meal value for animal feed.

Isothiocyanates

“Isothiocyanate” is a chemical group formed by substituting the oxygenin a isocyanate group with a sulfur. Isothiocyanate has the followingchemical structure:

Many natural isothiocyanates from plants are produced by conversion ofglucosinolates catalyzed by the enzyme myrosinase. Conversion ofsinigrin (allyl glucosinolate) catalyzed by myrosinase yields theproduct allyl isothiocyanate (also known as volatile oil of mustard).Allyl isothiocyanate is highly pungent and volatile, and is responsiblefor the pungent taste of horse radish and wasabi root. In pure form itcan be toxic, acting as an irritant to skin and mucous membranes. Allylisothiocyanate has the following chemical structure:

Myrosinase

“Myrosinase”, also known as thioglucosidase, is an enzyme that catalyzesthe conversion of glucosinolates to isothiocyanate compounds, which areextremely pungent and volatile. FIG. 1 shows the enzymatic reaction.Myrosinase first catalyzes the removal of D-glucose from glucosinolate,which results in an aglycone intermediate in the form ofthiohydroximate-O-sulfonate. Myrosinase then further catalyzes theremoval of the sulfate group to generate isothiocyanate.

While not the sole source of myrosinase, the seeds and other tissues ofplants in the Brassicaceae family are rich in myrosinase. Sinapis albais a particularly rich source of myrosinase. In a study carried out byAtle Bones (Bones, 1990), Sinapis alba seed was found to contain 10times more myrosinase than that of Brassica napus or Brassica rapa,while earlier work (Henderson and McEwen, 1972) demonstrated thatmyrosinase activity from crude extracts of Sinapis alba seeds greatlyexceeded that of other extract sources, including Brassica Juncea,Brassica nigra, Brassica napus, Brassica rapa and Crambe abyssinica.Other sources of myrosinase may include cresses, horseradish, wasabi,and camelina. However, any plant that expresses myrosinase inappreciable amounts, or organisms that are engineered to expressglucosinolate heterologously, such as bacteria, or plant and/ormammalian cell cultures, can potentially be used as a source ofmyrosinase enzyme of varying degrees of purity.

Myrosinase exists as several forms in Brassicaceae, and are encoded byat least three subfamilies of genes, denoted MA, MB, and MC, with thepotential for multiple genes in each subfamily However, in principle,any myrosinase will accept all glucosinolates as substrates (Rask,2000). Myrosinase is thought to be found exclusively in cells referredto as myrosin cells (reviewed in Kissen et al., 2009). Their relativelocalization with respect to glucosinolates is less well understood. Insome tissues, the enzyme and substrate are thought to be present in thesame cells but in different cellular compartments, while in othertissues, they are thought to segregate in different cell populations.Regardless of their relative distributions, it is thought that when theplant tissue is crushed or damaged, the two stores are brought together,bringing myrosinase in contact with glucosinolate and initiating theconversion of the glucosinolate to isothiocyanate. Thus, the myrosinasereaction serves the function of providing plants with a unique chemicaldefense against predation by herbivores

Oilseeds

“Oilseed” refers to any crop species where oil is extracted from theseeds of these grains for food or industrial purposes, and includesBrassicaceae oilseeds such as canola, and non-Brassicaceae oilseeds,such as flaxseed, soybean, safflower, and sunflower. An example of acrop species that produces a seed used primarily for the production ofedible oil is Brassica napus. An example of a crop species that is usedprimarily in the production of industrial feedstock oil is Brassicacarinata.

Brassica carinata is commonly referred to as Ethiopian Mustard, and is adomesticated oilseed Brassica species. Brassica carinata is anamphidiploid, meaning it is a stable hybrid between two diploid Brassicaprecursor species, specifically Brassica nigra and Brassica oleracea.The seed of Brassica carinata can contain more than 40% by weight ofoil, which can be used as an industrial feedstock as a replacement forpetroleum in a number applications.

Sinapis alba is commonly referred to as white mustard, and is an annualcrop of the Brassicaceae family The seed of Sinapis alba, used in thepreparation of condiment mustard, contains high concentrations ofglucosinolates, a sulfur containing compound that offers the plantprotection from insect predation.

Meal

“Meal” refers to the remaining fraction of the seed content afterextraction of the oil, and consists mainly of protein. In the case ofBrassica carinata this meal fraction is particularly protein richrelative to other Brassica oilseed species and has thus been proposed asa potential high value additive in animal feed applications. For thepresent invention, the meal to be treated can be derived from anyprocess the extracts oil from oilseeds, leaving the meal, including,without limitation hexane extraction and a cold press process.

Hexane Extraction to Extract the Oil from Oilseeds

Hexane extraction typically occurs by the following process. First, theoilseeds are prepared for the hexane extraction. The oilseeds arecleaned, and then crushed in a roller mill to generate flakes of0.3-0.38 mm in thickness. The flaked seed is then conveyed to a heateddrum where the flakes are cooked at elevated temperatures (from 80-150°C., depending on the source of the seed) for up to 20 min. Cooked seedflakes are then pressed in a series of screw presses or expellers whichcan remove 50-60% of the oil. Aside from the oil which is removed forfurther processing, the pressing produces a meal cake that is ideal forsolvent extraction.

“Meal cake” refers to the state of the seed meal after it has gonethrough the flaking and cooking stage and has been mechanically pressedto extrude the bulk of the oil. The term refers to the physicalcharacter of the meal at this stage, which has been compressed into acake-like mass rich in protein and still containing appreciable residualoil.

Next, using several cycles of countercurrent extraction, the meal cakeis treated with hexane to remove the residual oil from the meal. Afterthe oil has been removed from the flakes or meal cake, the meal willcontain approximately 30% of solvent (hexane) content.

“Defatted meal” refers to the state of the meal after the meal cake hasbeen solvent extracted to remove the last traces of residual oil.

Typically, the meal would then undergo toasting and desolventizing toremove the hexane solvent and reduce the moisture content to 12% orless. The meal is transferred to a desolventizer-toaster, where it isheated to remove remaining hexane. Most of the solvent is removed byheating the meal on steam-heated plates. Removal of the final traces ofsolvent is carried out by injecting steam through the meal (the actualtoasting process).

In the course of the toasting process (roughly 30 minutes in duration),the meal is exposed to temperatures ranging from 95-115° C. and moistureincreases to 12-18%. Subsequently, the meal is cooled and dried viaforced air circulation until a final moisture content of 12% or less isachieved. The meal is then pelletized or granulated depending on therequirements of the end user.

Cold Press Processing

In some cases the seed can also be processed using a cold pressmethodology, which is similar to above except it does not involve theuse of hexane to remove residual oil from the oil cake, resulting in ameal with much higher oil composition.

Process to Remove Glucosinolates from the Meal Fraction of Oilseeds

In one embodiment, the invention described herein provides a process forremoving at least one glucosinolate from a meal fraction of oilseedcomprising: (a) treating the meal fraction of oilseed with exogenousmyrosinase to convert the at least one glucosinolate to a volatileisothiocyanate; and (b) removing the volatile isothiocyanate from thetreated meal fraction of oilseed under conditions of mild heat andnegative pressure.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein step (a) and step (b) occur simultaneously.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein step (a) and step (b) occur sequentially.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the total time for step (a) and step (b) is greaterthan 2 min, 5 min, 10 min, 15 min, 20 min, 30 min, 1 h, 2 h, 3 h, or 4h,and less than 9h, 10h, 11 h, or 12 h.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the total time for step (a) and step (b) is 3 h.

In another embodiment, the processes described herein further comprisethe treatment of a seed meal cake containing glucosinolate wherein thetreatment involves incubating the meal with an exogenous source ofmyrosinase, and wherein the hydrolysis product is substantially removedfrom the seed meal cake by virtue of its high volatility.

Mild Heat

“Mild heat” means a temperature above 0° C., but less than a temperaturethat would have an adverse effect on protein quality in the meal becauseof heat denaturation. For example, mild heat can be greater than 25° C.but less than 90° C.

In one embodiment, the invention provides the process described hereinfor removing at least one glucosinolate from a meal fraction of oilseed,wherein the process further comprises regulating the temperature duringstep (a) and step (b) to prevent the temperature from exceeding 45° C.,50° C., 55° C., 60° C., 65° C., or 70° C.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the process further comprises regulating thetemperature during step (a) and step (b) to prevent the temperature fromexceeding 50° C.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the process further comprises regulating thetemperature during step (a) and step (b) to prevent the temperature fromexceeding 60° C.

Negative Pressure

“Negative Pressure” means any percentage of vacuum. For example,negative pressure can be anything from 0.0001% vacuum to 100% vacuum.

In one embodiment, the invention provides the process described hereinfor removing at least one glucosinolate from a meal fraction of oilseed,wherein the process further comprises carrying out step (a) and step (b)in a reaction vessel under negative pressure and evacuating volatilesubstances from the reaction vessel.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the process further comprises carrying out step (a) andstep (b) in a reaction vessel under negative pressure of over 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% vacuum andevacuating volatile substances from the reaction vessel.

Post-Treatment Incubation

Following the initial steps of treating the meal fraction of oilseedwith exogenous myrosinase to convert the at least one glucosinolate to avolatile isothiocyanate, and removing the volatile isothiocyanate fromthe treated meal fraction of oilseed under conditions of mild heat andnegative pressure, the meal fraction of oilseed can be further heated todry the meal and to remove additional volatile isothiocyanates.

In one embodiment, the invention provides the process described hereinfor removing at least one glucosinolate from a meal fraction of oilseed,wherein the process further comprises increasing the temperature to atemperature higher than that of step (b) following step (a) and step (b)and further incubating the meal fraction of oilseed until the moisturecontent of the meal fraction of oilseed decreases to the requiredpercentage.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the process further comprises increasing thetemperature to over 60° C., 65° C., 70° C., 75° C., 80° C., 85° C., or90° C., but to less than 95° C., 100° C., 105° C., 110° C., or 115° C.,following step (a) and step (b) and further incubating the meal fractionof oilseed until the moisture content of the meal fraction of oilseeddecreases to less than 20%, 18%, 16%, 14%, 12%, or 10%.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the process further comprises increasing thetemperature to 70° C. following step (a) and step (b) and furtherincubating the meal fraction of oilseed until the moisture content ofthe meal fraction of oilseed decreases to less than 20%, 18%, 16%, 14%,12%, or 10%.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the process further comprises increasing thetemperature to over 60° C., 65° C., 70° C., 75° C., 80° C., 85° C., or90° C., but to less than 95 ° C., 100° C., 105° C., 110° C., or 115° C.,following step (a) and step (b) and further incubating the meal fractionof oilseed until the moisture content of the meal fraction of oilseeddecreases to less than 12%.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the process further comprises increasing thetemperature to 70° C. following step (a) and step (b) and furtherincubating the meal fraction of oilseed until the moisture content ofthe meal fraction of oilseed decreases to less than 12%.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the process further comprises increasing thetemperature to over 60° C., 65° C., 70° C., 75° C., 80° C., 85° C., or90° C., but to less than 95 ° C., 100° C., 105° C., 110° C., or 115° C.,following step (a) and step (b) and further incubating the meal fractionof oilseed until the moisture content of the meal fraction of oilseeddecreases to less than 20%, 18%, 16%, 14%, 12%, or 10%, and wherein theprocess further comprises carrying out the post-treatment incubation ina reaction vessel under negative pressure and evacuating volatilesubstances from the reaction vessel.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the process further comprises increasing thetemperature to 70° C. following step (a) and step (b) and furtherincubating the meal fraction of oilseed until the moisture content ofthe meal fraction of oilseed decreases to less than 20%, 18%, 16%, 14%,12%, or 10%, and wherein the process further comprises carrying out thepost-treatment incubation in a reaction vessel under negative pressureand evacuating volatile substances from the reaction vessel.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the process further comprises increasing thetemperature to over 60° C., 65° C., 70° C., 75° C., 80° C., 85° C., or90° C., but to less than 95° C., 100° C., 105° C., 110° C., or 115° C.,following step (a) and step (b) and further incubating the meal fractionof oilseed until the moisture content of the meal fraction of oilseeddecreases to less than 12%, and wherein the process further comprisescarrying out the post-treatment incubation in a reaction vessel undernegative pressure and evacuating volatile substances from the reactionvessel.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the process further comprises increasing thetemperature to 70° C. following step (a) and step (b) and furtherincubating the meal fraction of oilseed until the moisture content ofthe meal fraction of oilseed decreases to less than 12%, and wherein theprocess further comprises carrying out the post-treatment incubation ina reaction vessel under negative pressure and evacuating volatilesubstances from the reaction vessel.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the process further comprises increasing thetemperature to over 60° C., 65° C., 70° C., 75° C., 80° C., 85° C., or90° C., but to less than 95° C., 100° C., 105° C., 110° C., or 115° C.,following step (a) and step (b) and further incubating the meal fractionof oilseed until the moisture content of the meal fraction of oilseeddecreases to less than 20%, 18%, 16%, 14%, 12%, or 10%, and wherein theprocess further comprises carrying out the post-treatment incubation ina reaction vessel under negative pressure of over 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% vacuum and evacuating volatilesubstances from the reaction vessel.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the process further comprises increasing thetemperature to 70° C. following step (a) and step (b) and furtherincubating the meal fraction of oilseed until the moisture content ofthe meal fraction of oilseed decreases to less than 20%, 18%, 16%, 14%,12%, or 10%, and wherein the process further comprises carrying out thepost-treatment incubation in a reaction vessel under negative pressureof over 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%vacuum and evacuating volatile substances from the reaction vessel.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the process further comprises increasing thetemperature to over 60° C., 65° C., 70° C., 75° C., 80° C., 85° C., or90° C., but to less than 95° C., 100° C., 105° C., 110° C., or 115° C.,following step (a) and step (b) and further incubating the meal fractionof oilseed until the moisture content of the meal fraction of oilseeddecreases to less than 12%, and wherein the process further comprisescarrying out the post-treatment incubation in a reaction vessel undernegative pressure of over 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, 98%, or 99% vacuum and evacuating volatile substances from thereaction vessel.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the process further comprises increasing thetemperature to 70° C. following step (a) and step (b) and furtherincubating the meal fraction of oilseed until the moisture content ofthe meal fraction of oilseed decreases to less than 12%, and wherein theprocess further comprises carrying out the post-treatment incubation ina reaction vessel under negative pressure of over 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% vacuum and evacuating volatilesubstances from the reaction vessel.

Mixing

During the initial steps of treating the meal fraction of oilseed withexogenous myrosinase to convert the at least one glucosinolate to avolatile isothiocyanate, and removing the volatile isothiocyanate fromthe treated meal fraction of oilseed under conditions of mild heat andnegative pressure, and/or the further incubation at higher heat, themeal fraction of oilseed and exogenous myrosinase can be continuouslymixed.

In one embodiment, the invention provides the process described hereinfor removing at least one glucosinolate from a meal fraction of oilseed,wherein the process further comprises continuously mixing the mealfraction of oilseed and exogenous myrosinase during step (a) and step(b).

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the process further comprises continuously mixing themeal fraction of oilseed and exogenous myrosinase during step (a), step(b), and the post-treatment incubation.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the process further comprises continuously mixing themeal fraction of oilseed and exogenous myrosinase during thepost-treatment incubation.

Preheating

Prior to the steps of treating the meal fraction of oilseed withexogenous myrosinase to convert the at least one glucosinolate to avolatile isothiocyanate, and removing the volatile isothiocyanate fromthe treated meal fraction of oilseed under conditions of mild heat andnegative pressure, the meal fraction of oilseed can be preheated.

In one embodiment, the invention provides the process described hereinfor removing at least one glucosinolate from a meal fraction of oilseed,wherein the process further comprises preheating the meal fraction ofoilseed to between 25° C. to 40° C. prior to step (a).

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the process further comprises preheating the mealfraction of oilseed to between 30° C. to 40° C. prior to step (a).

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the process further comprises preheating the mealfraction of oilseed to 35° C. prior to step (a).

Myrosinase

Any exogenous myrosinase source can be used to convert glucosinolateinto isothiocyanate. However, for reasons of economy, the source ofexogenous myrosinase does not have to be highly purified and can consistsimply of a slurry of readily available defatted meal in water, termed a“triggering solution”. Any defatted meal wherein the myrosinase is stillactive can be used as the source of myrosinase in the triggeringsolution, including, for example, the meal of oilseeds of the Brassicaceae family, including, for example, Sinapis alba and Brassicacarinata. The triggering solution may also comprise ascorbic acid. Thetriggering solution may be incubated at room temperature for a period oftime prior to its addition to the meal cake.

In another embodiment, the triggering solution is mixed with water andascorbic acid to form a slurry which is incubated at room temperaturefor a period of time subsequent to its addition to the meal cake.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the process further comprises providing the exogenousmyrosinase in the form of a triggering solution comprising a slurry ofdefatted meal in water.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the process further comprises providing the exogenousmyrosinase in the form of a triggering solution comprising a slurry ofdefatted meal in water, and wherein the defatted meal is derived fromoilseed of a plant species of the Brassicaceae family

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the process further comprises providing the exogenousmyrosinase in the form of a triggering solution comprising a slurry ofdefatted meal in water, and wherein the defatted meal is derived fromoilseed of Sinapis alba (White mustard; mustard seed flour).

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the process further comprises providing the exogenousmyrosinase in the form of a triggering solution comprising a slurry ofdefatted meal in water, and wherein the defatted meal is derived fromoilseed of Brassica carinata.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the process further comprises providing the exogenousmyrosinase in the form of a triggering solution comprising a slurry ofdefatted meal in water, and wherein the triggering solution furthercomprises ascorbic acid.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the process further comprises providing the exogenousmyrosinase in the form of a triggering solution comprising a slurry ofdefatted meal in water, wherein the triggering solution furthercomprises ascorbic acid, and wherein the defatted meal is present in apercent weight of 0.5%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 15%, or 20%, and the ascorbic acid is present in a percent weightof 0.001%, 0.005%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%,0.08%, 0.09%, or 0.1%.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the process further comprises providing the exogenousmyrosinase in the form of a triggering solution comprising a slurry ofdefatted meal in water, wherein the triggering solution furthercomprises ascorbic acid, and wherein the triggering solution comprisesthe water, defatted meal, and ascorbic acid in a ratio of 100:5:0.045w/w/w.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the process further comprises providing the exogenousmyrosinase in the form of a triggering solution comprising a slurry ofdefatted meal in water, and wherein the process further comprises mixingthe triggering solution for between 5 min to 30 min at room temperatureprior to treating the meal.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the process further comprises providing the exogenousmyrosinase in the form of a triggering solution comprising a slurry ofdefatted meal in water, wherein the triggering solution furthercomprises ascorbic acid, and wherein the process further comprisesmixing the triggering solution for between 5 min to 30 min at roomtemperature prior to treating the meal.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the process further comprises providing the exogenousmyrosinase in the form of a triggering solution comprising a slurry ofdefatted meal in water, and wherein the process further comprises mixingthe triggering solution for 15 min or 20 min at room temperature priorto treating the meal.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the process further comprises providing the exogenousmyrosinase in the form of a triggering solution comprising a slurry ofdefatted meal in water, wherein the triggering solution furthercomprises ascorbic acid, and wherein the process further comprisesmixing the triggering solution for 15 min or 20 min at room temperatureprior to treating the meal.

In a non-limiting, illustrative example, the defatted meal for thetriggering solution can be prepared by grinding the seed in a blender,for example a Waring™ blender, at medium speed into a uniform meal (i.e.until no full seeds remain visible). The contents of the blender arethen emptied into a metal sieving pan and placed on a heating plate setat between 40° C. to 90° C. for approximately 20 minutes. During thistime, the ground seeds are mixed occasionally to prevent charring. Thepan is then removed from the heat and allowed to cool at roomtemperature. Then, two volumes ground seed is mixed vigorously with onevolume hexane in a bottle. The phases are allowed to separate, and theoil phase is removed while the solid meal fraction is re-extracted threeadditional times with the same volume of hexane as described. After thefinal extraction, the meal is removed from the bottle and allowed toair-dry in a fume cabinet overnight. The triggering solution can then beprepared by adding water, or by adding water and ascorbic acid.

Adulteration of the meal by addition of toxic or noxious chemical agentsduring the course of removing glucosinolates from the meal will greatlyaffect the downstream usage of the processed meal. One advantage of thisprocess is that the triggering solution consists of only water, ascorbicacid and defatted meal, which are innocuous in terms of their potentialfor meal adulteration. In particular ascorbic acid has been shown to bea non-competitive activator of plant myrosinase and is required tomaintain the activity of myrosinase during the process. Ascorbic acid isan inexpensive, readily available, food quality component usedextensively in the food manufacturing and processing industry and doesnot detrimentally affect the quality of the processed meal. For example,Ascorbic Acid (Powder/USP/FCC) can be purchased from Fisher Chemical,catalogue number A62-212.

Buffering agents are not required for efficacy of the triggeringsolution. However, in one embodiment, the present invention provides theprocess described herein, wherein the triggering solution furthercomprises a buffer. In another embodiment, the present inventionprovides the process described herein, wherein the triggering solutionfurther comprises a buffer, and wherein the buffer comprises 0.1Mpotassium phosphate, pH 6.5.

In another embodiment, the present invention provides a process forpreparing the triggering solution comprising mixing mustard seed flour,water, and ascorbic acid in the ratio of 5 kg mustard seed flour to 100kg water to 45 g ascorbic acid to form a slurry, and incubating theslurry at room temperature for 20 minutes prior to addition to the mealcake.

In another embodiment, the present invention provides a process forpreparing the triggering solution comprising mixing 120 kg of soft waterand 45 g of ascorbic acid in a tank of suitable size, adding 5 kg ofdefatted Sinapis alba meal, and mixing the resultant slurry for 15minutes at room temperature.

In another embodiment, the present invention provides a process forpreparing the triggering solution comprising mixing 120 kg of soft waterand 45 g of ascorbic acid in a tank of suitable size, adding 5 kg ofdefatted Sinapis alba meal, and mixing the resultant slurry for 20minutes at room temperature.

In another embodiment, the present invention provides a process forpreparing the triggering solution comprising mixing 120 kg of soft waterand 45 g of ascorbic acid in a tank of suitable size, adding 5 kg ofdefatted Brassica carinata meal, and mixing the resultant slurry for 15minutes at room temperature.

In another embodiment, the present invention provides a process forpreparing the triggering solution comprising mixing 120 kg of soft waterand 45 g of ascorbic acid in a tank of suitable size, adding 5 kg ofdefatted Brassica carinata meal, and mixing the resultant slurry for 20minutes at room temperature.

Treating with Exogenous Myrosinase

Myrosinase can be added to the meal fraction of oilseed in any formatand amount that provides sufficient myrosinase activity to convert theglucosinolates in the meal fraction of oilseed to isothiocyanates withina reasonable time period.

In one embodiment, the invention provides the process described hereinfor removing at least one glucosinolate from a meal fraction of oilseed,wherein the process further comprises adding triggering solution to themeal fraction of oilseed to reach a final moisture content in the mealfraction of oilseed of 20% to 40%.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the process further comprises adding triggeringsolution to the meal fraction of oilseed to reach a final moisturecontent in the meal fraction of oilseed of 30%.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the process further comprises adding triggeringsolution to the meal fraction of oilseed in a ratio of 0.5:1, 0.55:1,0.6:1, 0.65:1, or 0.7:1 (w/w) triggering solution: meal fraction ofoilseed.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the process further comprises adding triggeringsolution to the meal fraction of oilseed in a ratio of 125:200 (w/w)triggering solution: meal fraction of oilseed.

Formation of the Meal Fraction of Oilseed

The meal fraction of oilseed to be processed by the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed can be the by-product of any process for extracting oil fromoilseeds.

In one embodiment, the invention provides the process described hereinfor removing at least one glucosinolate from a meal fraction of oilseed,wherein the meal to be treated consists of meal samples obtained from acommercial crushing and oil extraction process as defined above.

In another embodiment, the processes described herein can be applied toseed meals obtained from seeds of the Brassicaceae, based on treatmentof an oilseed meal cake, obtained after hexane extraction or derivedfrom a cold-press process.

(a) Hexane Extraction

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the meal fraction of oilseed results from hexaneextraction of oilseeds.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the meal fraction of oilseed results from hexaneextraction of oilseeds, and wherein the oilseeds were flaked and cookedprior to hexane extraction.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the meal fraction of oilseed results from hexaneextraction of oilseeds, wherein the oilseeds were flaked and cookedprior to hexane extraction, and wherein the cooking was carried out at atemperature greater than 70° C., 75° C., 80° C., 85° C., 90° C., 95° C.,100° C., 105° C., 110° C., 115° C., 120° C., 125° C., 130° C., 135° C.,or 140° C., but less than 150° C., 155° C., 160° C., 165° C., 170° C.,175° C., or 180° C.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the meal fraction of oilseed results from hexaneextraction of oilseeds, wherein the oilseeds were flaked and cookedprior to hexane extraction, and wherein the duration of cooking was atleast 10, 12, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, or 30 minutes,but less than 60, 70, 80, 90, or 100 minutes.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the meal fraction of oilseed results from hexaneextraction of oilseeds, wherein the oilseeds were flaked and cookedprior to hexane extraction, and wherein the flaked and cooked oilseedswere crushed to leave a meal cake with the bulk of the oil removed priorto hexane extraction.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the meal fraction of oilseed results from hexaneextraction of oilseeds, wherein the oilseeds were flaked and cookedprior to hexane extraction, and wherein the flaked and cooked oilseedswere crushed using an expeller or a screw press prior to hexaneextraction.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the meal fraction of oilseed results from hexaneextraction of oilseeds, wherein the oilseeds were flaked and cookedprior to hexane extraction, and wherein the flaked and cooked oilseedswere crushed using an expeller prior to hexane extraction.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the meal fraction of oilseed results from hexaneextraction of oilseeds, wherein the oilseeds were flaked and cookedprior to hexane extraction, and wherein the flaked and cooked oilseedswere crushed using a screw press prior to hexane extraction.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the meal fraction of oilseed results fromcountercurrent hexane extraction of oilseeds.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the meal fraction of oilseed results from hexaneextraction of oilseeds, and wherein the meal fraction of oilseed ishexane-extracted meal treated in a desolventizer-toaster (DT) to removethe residual hexane.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the meal fraction of oilseed results from hexaneextraction of oilseeds, wherein the meal fraction of oilseed ishexane-extracted meal treated in a desolventizer-toaster (DT) to removethe residual hexane, and wherein the treatment in the DT was at atemperature of at least 50° C., 55° C., 60° C., 65° C., 70° C., 75° C.,80° C., 85° C., 90° C., 95° C., 100° C., 105° C., 110° C., 115° C., 120°C., 125° C., or 130° C., but less than 135° C., 140° C., 145° C., or150° C.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the meal fraction of oilseed results from hexaneextraction of oilseeds, wherein the meal fraction of oilseed ishexane-extracted meal treated in a desolventizer-toaster (DT) to removethe residual hexane, and wherein the meal fraction of oilseed ishexane-extracted, desolventizer-toaster-treated meal dried to less than12% humidity.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the process further comprises producing the meal to betreated by hexane extracting oilseeds.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the process further comprises producing the meal to betreated by hexane extracting oilseeds, and flaking the oilseed prior tocooking for hexane extraction.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the process further comprises producing the meal to betreated by hexane extracting oilseeds, flaking the oilseed prior tocooking for hexane extraction, and cooking the flaked oilseed at atemperature greater than 70° C., 75° C., 80° C., 85° C., 90° C., 95° C.,100° C., 105° C., 110° C., 115° C., 120° C., 125° C., 130° C., 135° C.,or 140° C., but less than 150° C., 155° C., 160° C., 165° C., 170° C.,175° C., or 180° C. prior to hexane extraction.

In one such embodiment, the invention provides the process describedherein, wherein the process further comprises pretreating and flakingthe oilseeds at 85-95° C. prior to hexane extraction. In another suchembodiment, the invention provides the process described herein, whereinthe process further comprises pretreating and flaking the oilseeds at105° C. or higher prior to hexane extraction. In the latter embodiment,the glucosinolate levels of the meal cake may be reduced substantiallyfrom that of meal cake obtained from a process as outlined in the formerembodiment.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the process further comprises producing the meal to betreated by hexane extracting oilseeds, flaking the oilseed prior tocooking for hexane extraction, and cooking the flaked oilseed for atleast 10, 12, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28 or 30 minutes,but less than 60, 70, 80, 90, or 100 minutes prior to hexane extraction.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the process further comprises producing the meal to betreated by hexane extracting oilseeds, flaking the oilseed prior tocooking for hexane extraction, and crushing the flaked, cooked oilseedto leave a meal cake with the bulk of the oil removed prior to thehexane extraction.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the process further comprises producing the meal to betreated by hexane extracting oilseeds, flaking the oilseed prior tocooking for hexane extraction, and pressing the flaked and cookedoilseed through an expeller or a screw press prior to hexane extraction.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the process further comprises treating the meal cake bycountercurrent hexane extraction to remove remaining oil.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the process further comprises producing the meal to betreated by hexane extracting oilseeds, and treating the hexane-extractedmeal cake in a desolventizer-toaster (DT) to remove the residual hexaneprior to treating the meal with exogenous myrosinase.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the process further comprises producing the meal to betreated by hexane extracting oilseeds, and treating the hexane-extractedmeal cake in a desolventizer-toaster (DT) to remove the residual hexaneprior to treating the meal with exogenous myrosinase, and wherein thetreatment in the DT is at a temperature of at least 50° C., 55° C., 60°C., 65° C., 70° C., 75° C., 80° C., 85° C., 90° C., 95° C., 100° C.,105° C., 110° C., 115° C., 120° C., 125° C., 130° C., but less than 135°C., 140° C., 145° C., or 150° C.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the process further comprises producing the meal to betreated by hexane extracting oilseeds, treating the hexane-extractedmeal cake in a desolventizer-toaster (DT) to remove the residual hexaneprior to treating the meal with exogenous myrosinase, and drying thehexane-extracted, desolventizer-toaster-treated meal cake to less than12% humidity prior to treating the meal with exogenous myrosinase.

(b) Cold Press Processing

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the meal fraction of oilseed results from cold pressprocessing of oilseeds.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the process further comprises producing the mealfraction of oilseeds by a cold press process.

Preparing the Meal Fraction of Oilseeds for Processing

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the process further comprises carrying out thetreatment as a batch process on a meal cake, comprising glucosinolate.In such an embodiment, the meal cake should be of a consistency thatwould allow for optimal penetration of a triggering solution.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the meal fraction of oilseed to be treated is pelleted.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the meal fraction of oilseed to be treated is pelleted,and wherein the pelleted meal fraction of oilseed is homogenized byhammer-milling

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the meal fraction of oilseed to be treated is pelleted,wherein the pelleted meal fraction of oilseed is homogenized byhammer-milling, and wherein the homogenized meal fraction of oilseed hasa size of less than 5.66 mm, 4.75 mm, 4.00 mm, 3.36 mm, 2.83 mm or 2.38mm.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the meal fraction of oilseed to be treated is pelleted,wherein the pelleted meal fraction of oilseed is homogenized byhammer-milling, and wherein the homogenized meal fraction of oilseed hasa size of less than 3.36 mm.

In another embodiment, the present invention provides the processdescribed herein, wherein the meal to be treated is finely granularmeal. Finely granular meal can be prepared from pelleted meal byhomogenizing with a hammer mill (such as Model G5HFS1, serial number5075, Prater Industries, Chicago Ill.). The hammer mill can be equippedwith 8/64″ screens, which is standard equipment in a seed crushingplant.

In one such embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the process further comprises applying the processes topelleted meal that was processed in a hammer mill equipped with screensof 8/64″ to obtain a suitable consistency prior to application of thebatch process.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the process further comprises pelleting the mealfraction of oilseed prior to step (a).

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the process further comprises homogenizing the pelletedmeal with a hammer mill prior to step (a).

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the process further comprises homogenizing the pelletedmeal to a size of less than 5.66 mm, 4.75 mm, 4.00 mm, 3.36 mm, 2.83 mm,or 2.38 mm, prior to step (a).

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the process further comprises homogenizing the pelletedmeal to a size less than 3.36 mm, prior to step (a).

Oilseeds

The meal fraction of any oilseed that has glucosinolates can be used asthe substrate from the process described herein for removing at leastone glucosinolate from a meal fraction of oilseed.

In one embodiment, the invention provides the process described hereinfor removing at least one glucosinolate from a meal fraction of oilseed,wherein the meal fraction of oilseed is from oilseed of a plant speciesof the family Bras sicaceae.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the meal fraction of oilseed is from oilseed ofBrassica juncea, Brassica napus, Brassica rapa, Brassica carinata,Brassica nigra, Cammelina sativa, Crambe abyssinica, or Thlaspi arvense.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the meal fraction of oilseed is from oilseed ofBrassica carinata.

With respect to Brassica carinata under the reaction conditions citedabove, the added myrosinase converts sinigrin to allyl-isothiocyanate.In a novel aspect of this invention which relies on the low vaporpressure (high volatility) of allyl-isothiocyanate, the vesseltemperature is then adjusted and the vessel allowed to vent undernegative pressure, permitting the bulk of the volatileallyl-isothiocyanate to be removed from the vessel. As a result of suchtreatment, meal with substantially reduced glucosinolate content isderived as exemplified below.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the meal fraction of oilseed is from oilseed of Sinapisalba.

In another embodiment, the present invention provides the processdescribed herein, wherein the meal fraction of oilseeds is from Camelinasativa (false flax), Crambe abyssinica (Crambe) or Thlaspi arvense(pennycress).

In another embodiment, the present invention provides the processdescribed herein, wherein the meal fraction of oilseeds is from doublezero quality canola meal, and wherein the process reduces the existinglow levels of glucosinolates to non-detectable levels.

Reaction Vessel

In one embodiment, the invention provides the process described hereinfor removing at least one glucosinolate from a meal fraction of oilseed,wherein the process further comprises carrying out the treatment and/orpost-treatment incubation in a reaction vessel equipped to allow forcontinuous mixing of its contents, regulation of the internaltemperature of the vessel contents, and venting to allow for separatingand removing volatiles or gaseous products from the contents of thevessel.

In another embodiment, the processes described herein further comprise atreatment of a seed meal cake containing glucosinolate, wherein thetreatment involves incubating the meal with an exogenous source ofmyrosinase, wherein the hydrolysis product is substantially removed fromthe seed meal cake by virtue of its high volatility, and wherein theremoval of the isothiocyanate component of the reaction is facilitatedby application of reduced pressure to the reaction vessel.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the removal of glucosinolates from the meal isinitiated by addition of the triggering solution to the bulk of themeal. The granular meal is then added, along with the triggeringsolution, to a closed reaction vessel of suitable size andconfiguration, equipped with a plough type mixer, heating and coolingcapability to allow maintenance of internal temperatures through a rangeof 35° C. to 70° C., and exhaust ports that can allow the chamber to bevented under negative pressure.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed, wherein the reaction vessel is a desolventizer-toaster, andwherein step (a), step (b), and the further incubation are part of adesolventizer-toaster step of hexane extraction of oilseeds.

An example of a reaction vessel that can be used for the presentinvention is a Littleford™ vacuum dryer (Littleford Reactor™ 600 litermodel FKM600-D 2Z, serial number 5132, Littleford-Day Inc., Florence,Ky.).

FIG. 2 is a schematic diagram of a Littleford™ vacuum dryer apparatusthat can be used for the batchwise removal of glucosinolate oilseedmeal. In a non-limiting, illustrative example, two hundred kilograms ofoilseed meal is placed in reaction vessel 2. The plough mixer (notvisible), connected to its drive unit 36 by coupler 34, is started andthe meal is heated to 35° C. At the same time, 120 kg of soft water isloaded into the 200 liter stainless steel mixing vessel 4 and 45 g ofascorbic acid is added. After 5 minutes of mixing the water and ascorbicacid with agitator 6 driven by motor 8, 5 kg of defatted oilseed meal isadded to the mixing vessel 4 to form the triggering solution. Thetriggering solution is circulated via an inline mixer, comprising piping20 attached to a recirculation pump 22 downstream of drain 24, for 20minutes and then sprayed into the reaction vessel 2. The inline mixerand the mixing vessel 4 are attached to the reaction vessel 2 via a tee10, a ball valve 12, piping 14, spray tee 16, and two spray nozzles 18.The dispensing of the slurry comprising the triggering solution requiresa spray head that will not be clogged by the granular nature of theslurry.

At the top of the reaction vessel 2 is a hatch 26 through which thematerial (e.g. meal) is added to the chamber. The reaction vessel 2 alsohas a chamber access hatch 28, a discharge hatch 30 for removal of theprocessed material, and a bottom port 32, with a double valve systemwith a reducer in between, for sampling from the chamber.

At the right-hand side of the top of the reaction vessel 2, the reactionvessel 2 is attached to a bag housing 38 containing three bags 40. Thebags are filters that isolate the contents of the chamber from thevacuum source 46/condenser 44, allowing the vapour to go through butkeeping the dust and particulates confined to the reaction vessel side.Vacuum line 42 is attached to a condenser 44. The vacuum source 46 is asteam ejector (SE1 SIHI).

In one embodiment, the present invention provides the process describedherein, wherein the triggering solution is allowed to react with theoilseed for 3 hours in reaction vessel 2. It can be appreciated thatduring this time the temperature will rise during the course of thereaction, to achieve a temperature of 45° C. to 50° C. by the end of thethree hour incubation. During this time the bulk of the glucosinolate isconverted to isothiocyanate. At the completion of the reaction, thetemperature in the reaction vessel 2 is raised to 60° C. to 70° C. toallow for drying of the meal to less than 12% retained humidity.

In another embodiment, the present invention provides the processdescribed herein, wherein vacuum is applied to the reaction vessel 2 andthe meal is then heated to 50° C. for an additional one hour. After theone hour hold at 50° C., the meal is heated to 70° C. to 74° C. anddried to a moisture content of <12%.

In another embodiment, the present invention provides the processdescribed herein, wherein vacuum is applied to reaction vessel 2 fromthe onset of the initial treatment.

In another embodiment, the present invention provides the processdescribed herein, wherein the contents of the reaction vessel 2 areinitially adjusted to 35° C., prior to the addition of the triggeringsolution.

The myrosinase catalyzed hydrolysis of glucosinolate is a highlyexothermic reaction and therefore once the reaction has commenced (byspraying the triggering solution onto the meal sample), cooling water iscirculated in the jacket (not visible) of the reaction vessel 2 to allowfor dissipation of the heat produced during the reaction. The reactionis allowed to proceed for a period of time sufficient to allow forsubstantial conversion of the glucosinolate to isothiocyanate.

In one embodiment, the present invention provides the process describedherein, wherein cooling water is circulated in the jacket (not visible)of reaction vessel 2 from the outset of the reaction to allow attainmentof a maximum temperature at 50° C.-60° C. during the three hourreaction.

In another embodiment, the present invention provides the processdescribed herein, wherein cooling water is circulated in the jacket (notvisible) of reaction vessel 2 only once the temperature in the chamberhad reached 35° C. during the initial reaction.

In another embodiment, the present invention provides the processdescribed herein, wherein the meal undergoing the process is obtainedfrom Brassica carinata, which contains sinigrin (2-propylglucosinolate)as the predominant glucosinolate species, which is converted to allylisothiocyanate, a volatile, extremely pungent and potent lachrymator, bythe action of myrosinase. In the examples provided, volatile allylisothiocyanate released by the reaction is vented to the exterior of thereaction vessel 2. A steam ejector vacuum source 46 applied to thereaction vessel 2 while venting allows for improved efficiency of allylisothiocyanate removal from the vessel during the three hour course ofthe reaction and subsequent meal drying step. An advantage ofmaintaining the vacuum is that the negative pressure coupled with therising temperature in the chamber and the higher temperatures during thedrying step, leads to more complete removal of the produced allylisothiocyanate than would normally be achievable, thus eliminating therequirement for auxiliary means of isothiocyanate removal, such as thoseoutlined previously, which can adversely affect the quality of theprocessed meal. As a result of such treatment, meal with substantiallyreduced glucosinolate content is derived as exemplified below. Thesubstantial reduction in glucosinolate may result in the meal havingless than 80% of their starting levels of glucosinolates.

In a non-limiting, illustrative example, the present invention providesa process comprising preparing a triggering solution of mustard seedflour, water and ascorbic acid in the ratio of 5 kg mustard seed flourto 100 kg water to 45 g ascorbic acid, and incubating the slurry at roomtemperature for 20 minutes prior to addition the meal cake; spraying thetriggering solution onto hammer milled meal contained in a reactionvessel of suitable size and capable of both heating and mixing as wellas venting to allow for removal of volatiles (e.g. a Littleford™ vacuumdryer); and incubating with continuous mixing at an appropriatetemperature and for an appropriate time.

Inline Removal of Glucosinolate During the Desolventizer-Toaster Step ofHexane Extraction

While the examples cited herein describe a process which is adapted tobatch processing of meal samples, the process can be adapted so that itcan function “inline” of an existing oilseed crushing/hexane extractionprocessing plant such as that described earlier. In particular, there isample opportunity to incorporate the process with very little disruptionor cost into the desolventizer-toaster stage, which can readily beadapted for the mixing, temperature regulation and venting described forthe batch glucosinolate removal process.

In one embodiment, the process described herein is incorporated into anexisting seed crushing line, to provide a continuous process such as wasdescribed in the background section.

In one embodiment, the invention provides a process for removing atleast one glucosinolate from a meal fraction of oilseed at thedesolventizer-toaster step of a hexane extraction process, comprising:(a) treating the meal fraction of oilseed with exogenous myrosinase, toconvert the at least one glucosinolate to volatile isothiocyanate; and(b) removing the volatile isothiocyanate from the treated meal fractionof oilseed under conditions of mild heat and negative pressure duringdesolventizing and toasting.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed at the desolventizer-toaster step of a hexane extractionprocess, wherein step (a) and step (b) occur simultaneously.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed at the desolventizer-toaster step of a hexane extractionprocess, wherein step (a) and step (b) occur sequentially.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed at the desolventizer-toaster step of a hexane extractionprocess, wherein the process further comprises continuously mixing themeal fraction of oilseed and exogenous myrosinase during step (a) andstep (b).

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed at the desolventizer-toaster step of a hexane extractionprocess, wherein the desolventizing and toasting time of step (b) isgreater than 2 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35min, 40 min, 50 min, or 1 h, but less than 1.5 h, 2h, 3h, 4h, or 5 h.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed at the desolventizer-toaster step of a hexane extractionprocess, wherein the desolventizing and toasting time of step (b) is 20min.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed at the desolventizer-toaster step of a hexane extractionprocess, wherein the desolventizing and toasting time of step (b) is 30min.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed at the desolventizer-toaster step of a hexane extractionprocess, wherein the process further comprises drying the meal fractionof oilseed until the moisture content of the meal decreases to less than20%, 18%, 16%, 14%, 12% or 10%.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed at the desolventizer-toaster step of a hexane extractionprocess, wherein the process further comprises drying the meal fractionof oilseed until the moisture content of the meal decreases to less than12%.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed at the desolventizer-toaster step of a hexane extractionprocess, wherein the process further comprises carrying out thedesolventizing and toasting in a desolventizer-toaster under negativepressure and evacuating volatile substances from thedesolventizer-toaster.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed at the desolventizer-toaster step of a hexane extractionprocess, wherein the desolventizer-toaster is under negative pressure ofover 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%vacuum.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed at the desolventizer-toaster step of a hexane extractionprocess, wherein the process further comprises providing the exogenousmyrosinase in the form of a triggering solution comprising a slurry ofdefatted meal in water.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed at the desolventizer-toaster step of a hexane extractionprocess, wherein the process further comprises providing the exogenousmyrosinase in the form of a triggering solution comprising a slurry ofdefatted meal in water, and wherein the defatted meal is from oilseed ofa plant species of the Bras sicaceae family

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed at the desolventizer-toaster step of a hexane extractionprocess, wherein the process further comprises providing the exogenousmyrosinase in the form of a triggering solution comprising a slurry ofdefatted meal in water, and wherein the defatted meal is from oilseed ofSinapis alba.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed at the desolventizer-toaster step of a hexane extractionprocess, wherein the process further comprises providing the exogenousmyrosinase in the form of a triggering solution comprising a slurry ofdefatted meal in water, and wherein the defatted meal is from oilseed ofBrassica carinata.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed at the desolventizer-toaster step of a hexane extractionprocess, wherein the process further comprises providing the exogenousmyrosinase in the form of a triggering solution comprising a slurry ofdefatted meal in water, and wherein the triggering solution furthercomprises ascorbic acid.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed at the desolventizer-toaster step of a hexane extractionprocess, wherein the process further comprises providing the exogenousmyrosinase in the form of a triggering solution comprising a slurry ofdefatted meal in water, wherein the triggering solution furthercomprises ascorbic acid, and wherein the defatted meal is present in apercent weight of 0.5%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 15%, or 20%, and the ascorbic acid is present in a percent weightof 0.001%, 0.005%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%,0.08%, 0.09%, or 0.1%.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed at the desolventizer-toaster step of a hexane extractionprocess, wherein the process further comprises providing the exogenousmyrosinase in the form of a triggering solution comprising a slurry ofdefatted meal in water, and wherein the triggering solution comprisesthe water, defatted meal, and ascorbic acid in a ratio of 100:5:0.045w/w/w.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed at the desolventizer-toaster step of a hexane extractionprocess, wherein the process further comprises providing the exogenousmyrosinase in the form of a triggering solution comprising a slurry ofdefatted meal in water, and wherein the process further comprises mixingthe triggering solution for between 5 min to 30 min at room temperatureprior to treating the meal fraction of oilseed.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed at the desolventizer-toaster step of a hexane extractionprocess, wherein the process further comprises providing the exogenousmyrosinase in the form of a triggering solution comprising a slurry ofdefatted meal in water, and wherein the process further comprises addingtriggering solution to the meal fraction of oilseed to reach a finalmoisture content in the meal fraction of oilseed of 20% to 40%.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed at the desolventizer-toaster step of a hexane extractionprocess, wherein the process further comprises providing the exogenousmyrosinase in the form of a triggering solution comprising a slurry ofdefatted meal in water, and wherein the process further comprises addingtriggering solution to the meal fraction of oilseed to reach a finalmoisture content in the meal fraction of oilseed of 30%.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed at the desolventizer-toaster step of a hexane extractionprocess, wherein the process further comprises providing the exogenousmyrosinase in the form of a triggering solution comprising a slurry ofdefatted meal in water, and wherein the process further comprises addingtriggering solution to the meal fraction of oilseed in a ratio of 0.5:1,0.55:1, 0.6:1, 0.65:1, or 0.7:1 (w/w) triggering solution: meal fractionof oilseed.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed at the desolventizer-toaster step of a hexane extractionprocess, wherein the process further comprises providing the exogenousmyrosinase in the form of a triggering solution comprising a slurry ofdefatted meal in water, and wherein the process further comprises addingtriggering solution to the meal fraction of oilseed in a ratio of125:200 (w/w) triggering solution: meal fraction of oilseed.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed at the desolventizer-toaster step of a hexane extractionprocess, wherein the oilseeds were flaked and cooked prior to hexaneextraction.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed at the desolventizer-toaster step of a hexane extractionprocess, wherein the oilseeds were flaked and cooked prior to hexaneextraction, and wherein the cooking was carried out at a temperaturegreater than 70° C., 75° C., 80° C., 85° C., 90° C., 95° C., 100° C.,105° C., 110° C., 115° C., 120° C., 125° C., 130° C., 135° C., or 140°C., but less than 150° C., 155° C., 160° C., 165° C., 170° C., 175° C.,or 180° C.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed at the desolventizer-toaster step of a hexane extractionprocess, wherein the oilseeds were flaked and cooked prior to hexaneextraction, and wherein the duration of cooking was at least 10, 12, 14,15, 16, 17, 18, 19, 20, 22, 24, 26, 28, or 30 minutes, but less than 60,70, 80, 90, or 100 minutes.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed at the desolventizer-toaster step of a hexane extractionprocess, wherein the hexane extraction was carried out on flaked andcooked oilseed that had been crushed using an expeller or a screw press.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed at the desolventizer-toaster step of a hexane extractionprocess, wherein the hexane extraction was carried out on flaked andcooked oilseed that had been crushed using an expeller.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed at the desolventizer-toaster step of a hexane extractionprocess, wherein the hexane extraction was carried out on flaked andcooked oilseed that had been crushed using a screw press.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed at the desolventizer-toaster step of a hexane extractionprocess, wherein the oilseeds were flaked and cooked prior to hexaneextraction, and wherein the flaked, cooked oilseeds were crushed toleave a meal cake with the bulk of the oil removed prior to hexaneextraction.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed at the desolventizer-toaster step of a hexane extractionprocess, wherein the meal fraction of oilseed results fromcountercurrent hexane extraction of oilseeds.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed at the desolventizer-toaster step of a hexane extractionprocess, wherein the meal fraction of oilseed is from oilseed of a plantspecies of the family Brassicaceae.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed at the desolventizer-toaster step of a hexane extractionprocess, wherein the meal fraction of oilseed is from oilseed ofBrassica juncea, Brassica napus, Brassica rapa, Brassica carinata,Brassica nigra, Cammelina sativa, Crambe abyssinica, or Thlaspi arvense.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed at the desolventizer-toaster step of a hexane extractionprocess, wherein the meal fraction of oilseed is from oilseed ofBrassica carinata.

In another embodiment, the invention provides the process describedherein for removing at least one glucosinolate from a meal fraction ofoilseed at the desolventizer-toaster step of a hexane extractionprocess, wherein the meal fraction of oilseed is from oilseed of Sinapisalba.

Removal of Glucosinolates from Brassica carinata Using Heat and Pressure

Brassica carinata seed is unique, because it has a lower fibre and ahigher protein content than other Brassica seeds, and also has a thinnerseed coat. In addition, current commercial varieties of Brassicacarinata seed contain different levels and types of glucosinates thanother members of the Brassicaceae family For example, Brassica carinataseed has higher overall levels of glucosinates, with the predominantform being sinigrin, than Brassica napus, which has little or nosinigrin (Xin et al., 2014). Therefore, Brassica carinata seed willprocess differently than other Brassica seeds, and it is not predictablehow Brassica carinata seeds will respond to techniques used on otherBrassica species.

In another embodiment, the present invention provides a process forremoving at least one glucosinolate from a meal fraction of a Brassicacarinata oilseed comprising heating and applying pressure to the oilseedbefore, during, or after the extraction of oil.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of a Brassica carinata oilseed comprising heating and applyingpressure to the oilseed before, during, or after the extraction of oil,wherein the temperature during heating is greater than 70° C., 75° C.,80° C., 85° C., 90° C., 95° C., 100° C., 105° C., 110° C., 115° C., 120°C., 125° C., 130° C., 135° C., or 140° C., but less than 150° C., 155°C., 160° C., 165° C., 170° C., 175° C., or 180° C.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of a Brassica carinata oilseed comprising heating and applyingpressure to the oilseed before, during, or after the extraction of oil,wherein the duration of heating is at least 10, 12, 14, 15, 16, 17, 18,19, 20, 22, 24, 26, 28, or 30 minutes, but less than 60, 70, 80, 90, or100 minutes.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of a Brassica carinata oilseed comprising heating and applyingpressure to the oilseed before, during, or after the extraction of oil,wherein the process further comprises applying the pressure using anexpeller or a screw press.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of a Brassica carinata oilseed comprising heating and applyingpressure to the oilseed before, during, or after the extraction of oil,wherein the process further comprises applying the pressure using anexpeller.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of a Brassica carinata oilseed comprising heating and applyingpressure to the oilseed before, during, or after the extraction of oil,wherein the process further comprises applying the pressure using ascrew press.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of a Brassica carinata oilseed comprising heating and applyingpressure to the oilseed before, during, or after the extraction of oil,wherein the process results in a reduction of glucosinolates of greaterthan 50%, 55%, 60%, 65%, 70%, or 75%.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of a Brassica carinata oilseed comprising heating and applyingpressure to the oilseed before, during, or after the extraction of oil,wherein the meal proteins are substantially preserved.

In another embodiment, the present invention provides a process forremoving at least one glucosinolate from a meal fraction of a Brassicacarinata oilseed comprising: (a) heating and applying pressure to theoilseed before, during, or after the extraction of oil; (b) treating themeal fraction of oilseed with exogenous myrosinase to convert the atleast one glucosinolate to a volatile isothiocyanate; and (c) removingthe volatile isothiocyanate from the treated meal fraction of oilseedunder conditions of mild heat and negative pressure.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of a Brassica carinata oilseed comprising: (a) heating andapplying pressure to the oilseed before, during, or after the extractionof oil; (b) treating the meal fraction of oilseed with exogenousmyrosinase to convert the at least one glucosinolate to a volatileisothiocyanate; and (c) removing the volatile isothiocyanate from thetreated meal fraction of oilseed under conditions of mild heat andnegative pressure; wherein the temperature of heating in step (a) isgreater than 70° C., 75° C., 80° C., 85° C., 90° C., 95° C., 100° C.,105° C., 110° C., 115° C., 120° C., 125° C., 130° C., 135° C., or 140°C., but less than 150° C., 155° C., 160° C., 165° C., 170° C., 175° C.,or 180° C.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of a Brassica carinata oilseed comprising: (a) heating andapplying pressure to the oilseed before, during, or after the extractionof oil; (b) treating the meal fraction of oilseed with exogenousmyrosinase to convert the at least one glucosinolate to a volatileisothiocyanate; and (c) removing the volatile isothiocyanate from thetreated meal fraction of oilseed under conditions of mild heat andnegative pressure; wherein the duration of heating in step (a) is atleast 10, 12, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, or 30 minutes,but less than 60, 70, 80, 90, or 100 minutes.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of a Brassica carinata oilseed comprising: (a) heating andapplying pressure to the oilseed before, during, or after the extractionof oil; (b) treating the meal fraction of oilseed with exogenousmyrosinase to convert the at least one glucosinolate to a volatileisothiocyanate; and (c) removing the volatile isothiocyanate from thetreated meal fraction of oilseed under conditions of mild heat andnegative pressure; wherein the process further comprises applying thepressure in step (a) using an expeller or a screw press.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of a Brassica carinata oilseed comprising: (a) heating andapplying pressure to the oilseed before, during, or after the extractionof oil; (b) treating the meal fraction of oilseed with exogenousmyrosinase to convert the at least one glucosinolate to a volatileisothiocyanate; and (c) removing the volatile isothiocyanate from thetreated meal fraction of oilseed under conditions of mild heat andnegative pressure; wherein the process further comprises applying thepressure in step (a) using an expeller.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of a Brassica carinata oilseed comprising: (a) heating andapplying pressure to the oilseed before, during, or after the extractionof oil; (b) treating the meal fraction of oilseed with exogenousmyrosinase to convert the at least one glucosinolate to a volatileisothiocyanate; and (c) removing the volatile isothiocyanate from thetreated meal fraction of oilseed under conditions of mild heat andnegative pressure; wherein the process further comprises applying thepressure in step (a) using a screw press.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of a Brassica carinata oilseed comprising: (a) heating andapplying pressure to the oilseed before, during, or after the extractionof oil; (b) treating the meal fraction of oilseed with exogenousmyrosinase to convert the at least one glucosinolate to a volatileisothiocyanate; and (c) removing the volatile isothiocyanate from thetreated meal fraction of oilseed under conditions of mild heat andnegative pressure; wherein step (b) and step (c) occur simultaneously orsequentially.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of a Brassica carinata oilseed comprising: (a) heating andapplying pressure to the oilseed before, during, or after the extractionof oil; (b) treating the meal fraction of oilseed with exogenousmyrosinase to convert the at least one glucosinolate to a volatileisothiocyanate; and (c) removing the volatile isothiocyanate from thetreated meal fraction of oilseed under conditions of mild heat andnegative pressure; wherein the time for step (b) and step (c) is greaterthan 2 min, 5 min, 10 min, 15 min, 20 min, 30 min, 1 h, 2 h, 3 h, or 4h, and less than 9 h, 10 h, 11 h, or 12 h.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of a Brassica carinata oilseed comprising: (a) heating andapplying pressure to the oilseed before, during, or after the extractionof oil; (b) treating the meal fraction of oilseed with exogenousmyrosinase to convert the at least one glucosinolate to a volatileisothiocyanate; and (c) removing the volatile isothiocyanate from thetreated meal fraction of oilseed under conditions of mild heat andnegative pressure; wherein the process further comprises regulating thetemperature during step (b) and step (c) to prevent the temperature fromexceeding 45° C., 50° C., 55° C., 60° C., 65° C., or 70° C.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of a Brassica carinata oilseed comprising: (a) heating andapplying pressure to the oilseed before, during, or after the extractionof oil; (b) treating the meal fraction of oilseed with exogenousmyrosinase to convert the at least one glucosinolate to a volatileisothiocyanate; and (c) removing the volatile isothiocyanate from thetreated meal fraction of oilseed under conditions of mild heat andnegative pressure; wherein the process further comprises carrying outstep (b) and step (c) in a reaction vessel under negative pressure ofover 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%vacuum and evacuating volatile substances from the reaction vessel.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of a Brassica carinata oilseed comprising: (a) heating andapplying pressure to the oilseed before, during, or after the extractionof oil; (b) treating the meal fraction of oilseed with exogenousmyrosinase to convert the at least one glucosinolate to a volatileisothiocyanate; and (c) removing the volatile isothiocyanate from thetreated meal fraction of oilseed under conditions of mild heat andnegative pressure; wherein the process further comprises increasing thetemperature to over 60° C., 65° C., 70° C., 75° C., 80° C., 85° C., or90° C., but to less than 95 ° C., 100° C., 105° C., 110° C., or 115° C.,following step (b) and step (c) and further incubating the meal fractionof oilseed until the moisture content of the meal fraction of oilseeddecreases to less than 20%, 18%, 16%, 14%, 12%, or 10%.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of a Brassica carinata oilseed comprising: (a) heating andapplying pressure to the oilseed before, during, or after the extractionof oil; (b) treating the meal fraction of oilseed with exogenousmyrosinase to convert the at least one glucosinolate to a volatileisothiocyanate; and (c) removing the volatile isothiocyanate from thetreated meal fraction of oilseed under conditions of mild heat andnegative pressure; wherein the process further comprises increasing thetemperature to over 60° C., 65° C., 70° C., 75° C., 80° C., 85° C., or90° C., but to less than 95° C., 100° C., 105° C., 110° C., or 115° C.,following step (b) and step (c) and further incubating the meal fractionof oilseed until the moisture content of the meal fraction of oilseeddecreases to less than 20%, 18%, 16%, 14%, 12%, or 10%; and wherein theprocess further comprises carrying out the further incubation in areaction vessel under negative pressure of over 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 98%, or 99% vacuum and evacuating volatilesubstances from the reaction vessel.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of a Brassica carinata oilseed comprising: (a) heating andapplying pressure to the oilseed before, during, or after the extractionof oil; (b) treating the meal fraction of oilseed with exogenousmyrosinase to convert the at least one glucosinolate to a volatileisothiocyanate; and (c) removing the volatile isothiocyanate from thetreated meal fraction of oilseed under conditions of mild heat andnegative pressure; wherein the process further comprises continuouslymixing the meal fraction of oilseed and exogenous myrosinase.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of a Brassica carinata oilseed comprising: (a) heating andapplying pressure to the oilseed before, during, or after the extractionof oil; (b) treating the meal fraction of oilseed with exogenousmyrosinase to convert the at least one glucosinolate to a volatileisothiocyanate; and (c) removing the volatile isothiocyanate from thetreated meal fraction of oilseed under conditions of mild heat andnegative pressure; wherein the process further comprises preheating themeal fraction of oilseed to between 25° C. to 40° C. prior to step (b).

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of a Brassica carinata oilseed comprising: (a) heating andapplying pressure to the oilseed before, during, or after the extractionof oil; (b) treating the meal fraction of oilseed with exogenousmyrosinase to convert the at least one glucosinolate to a volatileisothiocyanate; and (c) removing the volatile isothiocyanate from thetreated meal fraction of oilseed under conditions of mild heat andnegative pressure; wherein the process further comprises providing theexogenous myrosinase in the form of a triggering solution comprising aslurry of defatted meal in water.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of a Brassica carinata oilseed comprising: (a) heating andapplying pressure to the oilseed before, during, or after the extractionof oil; (b) treating the meal fraction of oilseed with exogenousmyrosinase to convert the at least one glucosinolate to a volatileisothiocyanate; and (c) removing the volatile isothiocyanate from thetreated meal fraction of oilseed under conditions of mild heat andnegative pressure; wherein the process further comprises providing theexogenous myrosinase in the form of a triggering solution comprising aslurry of defatted meal in water; and wherein the defatted meal is fromoilseed of a plant species of the Brassicaceae family

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of a Brassica carinata oilseed comprising: (a) heating andapplying pressure to the oilseed before, during, or after the extractionof oil; (b) treating the meal fraction of oilseed with exogenousmyrosinase to convert the at least one glucosinolate to a volatileisothiocyanate; and (c) removing the volatile isothiocyanate from thetreated meal fraction of oilseed under conditions of mild heat andnegative pressure; wherein the process further comprises providing theexogenous myrosinase in the form of a triggering solution comprising aslurry of defatted meal in water; and wherein the defatted meal is fromoilseed of Sinapis alba or Brassica carinata.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of a Brassica carinata oilseed comprising: (a) heating andapplying pressure to the oilseed before, during, or after the extractionof oil; (b) treating the meal fraction of oilseed with exogenousmyrosinase to convert the at least one glucosinolate to a volatileisothiocyanate; and (c) removing the volatile isothiocyanate from thetreated meal fraction of oilseed under conditions of mild heat andnegative pressure; wherein the process further comprises providing theexogenous myrosinase in the form of a triggering solution comprising aslurry of defatted meal in water; and wherein the triggering solutionfurther comprises ascorbic acid.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of a Brassica carinata oilseed comprising: (a) heating andapplying pressure to the oilseed before, during, or after the extractionof oil; (b) treating the meal fraction of oilseed with exogenousmyrosinase to convert the at least one glucosinolate to a volatileisothiocyanate; and (c) removing the volatile isothiocyanate from thetreated meal fraction of oilseed under conditions of mild heat andnegative pressure; wherein the process further comprises providing theexogenous myrosinase in the form of a triggering solution comprising aslurry of defatted meal in water; and wherein the process furthercomprises adding triggering solution to the meal fraction of oilseed ina ratio of 0.5:1, 0.55:1, 0.6:1, 0.65:1, or 0.7:1 (w/w) triggeringsolution: meal fraction of oilseed.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of a Brassica carinata oilseed comprising: (a) heating andapplying pressure to the oilseed before, during, or after the extractionof oil; (b) treating the meal fraction of oilseed with exogenousmyrosinase to convert the at least one glucosinolate to a volatileisothiocyanate; and (c) removing the volatile isothiocyanate from thetreated meal fraction of oilseed under conditions of mild heat andnegative pressure; wherein the meal fraction of oilseed to be treated insteps (b) and (c) is pelleted and homogenized by hammer-milling to asize of less than 5.66 mm, 4.75 mm, 4.00 mm, 3.36 mm, 2.83 mm or 2.38mm.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of a Brassica carinata oilseed comprising: (a) heating andapplying pressure to the oilseed before, during, or after the extractionof oil; (b) treating the meal fraction of oilseed with exogenousmyrosinase to convert the at least one glucosinolate to a volatileisothiocyanate; and (c) removing the volatile isothiocyanate from thetreated meal fraction of oilseed under conditions of mild heat andnegative pressure; wherein the process results in a reduction ofglucosinolates of greater than 75%, 80%, 85%, 90%, or 95%.

In another embodiment, the present invention provides the processdescribed herein for removing at least one glucosinolate from a mealfraction of a Brassica carinata oilseed comprising: (a) heating andapplying pressure to the oilseed before, during, or after the extractionof oil; (b) treating the meal fraction of oilseed with exogenousmyrosinase to convert the at least one glucosinolate to a volatileisothiocyanate; and (c) removing the volatile isothiocyanate from thetreated meal fraction of oilseed under conditions of mild heat andnegative pressure; wherein the meal proteins are substantiallypreserved.

EXAMPLES Example 1 Analytical Methods for Determination of Glucosinolate(GSL) and Allyl-Isothiocyanate

An HPLC analytical method was adapted from two publications (Cools andTerry, 2012; Berhow et al., 2013). Modifications included mobile phasecomposition and gradient conditions to allow for analysis of sinigrinand its allyl isothiocyanate degradation product in a single injection.The analytical system was comprised of an Agilent™ 110 HPLC systemequipped with a binary pump, autosampler, column compartment andvariable wavelength detector, and Chemstation™ control and dataacquisition software. The HPLC column utilized was a Waters Xterra™RP18, 3.5 μm, 4.6×250 mm analytical column and a 20 μL injection volumewas used. The detection wavelengths used were 226 nm for sinigrin and242 nm for allyl-isothiocyanate. Reference standards used were sinigrin(Sigma™ S1647) and allyl-isothiocyanate (Sigma™ 36682). The mobilephases used were Acetonitrile (Phase A) and DI H2O (Phase B). Solventdelivery used a gradient profile as follows; 0-2.5min 99%B, 3-7.5min30%B, 8.5-10min 99%B with a 1.5 min post-run hold. To investigate thelinearity of response, calibrators for sinigrin and allyl-isothiocyanatewere prepared at 6 concentrations covering a concentration range from5-1000 μg/mL (shown below).

Analyte Retention Time (min) Concentration (μg/mL) Peak Area Sinigrin3.10 5 41.07 10 90.24 50 430.26 100 839.18 500 4129.71 1000 8284.68 AITC9.14 5 22.07 10 59.66 50 218.61 100 408.09 500 1893.30 1000 3948.86

Injection of 3 separate preparations of the 6-point calibration curvesshowed variability of less than 10% for both retention time and peakarea. Conditions for extraction of sinigrin from seed meals weredetermined and optimized and the extracts showed no significantinterference at the retention times for either anylate and did notaffect peak shape. Extraction parameters developed were 50% aqueousacetonitrile added in a 20:1 ratio to seed meal (e.g. 10 mL solvent to500 mg seed meal) and placed in boiling water bath in sealed containersfor 30 min. The resulting samples were centrifuged, supernatant diluted1:1 with DI H2O and then again 1:1 with 75% aqueous acetonitrile, andthe final solution filtered prior to injection.

Example 2 Preparation of Exogenous Myrosinase Source

Defatted meal from seeds of Sinapis alba c.v. Andante or Brassicacarinata A100 was prepared as follows: 4.5 kg of clean seed was groundusing a Waring™ blender at medium speed into a uniform meal (i.e. untilno full seeds remained visible). The contents of the blender was emptiedinto a metal sieving pan and placed on a heating plate set at 80° C. for20 minutes. During this time the ground seeds were mixed occasionally toprevent charring. The pan was then removed from the heat and allowed tocool at room temperature. 400 mL of ground seed was then placed in a oneliter bottle, 200 mL hexane was added and then the capped bottle washand shaken vigorously. The phases were allowed to separate and the oilphase was removed while the solid meal fraction was re-extracted threeadditional times with 200 ml hexane as described. After the finalextraction, the meal was removed from the bottle and allowed to air-dryin a fume cabinet overnight. The relative myrosinase activity of thedefatted meal samples processed as above was estimated by incubatingmeal samples in the presence of exogenous sinigrin for 3, 5, 10 and 15minutes, extracting the sinigrin and allyl-isothiocyanate as describedin Example 1, and measuring allyl isothiocyanate production viaestimation of HPLC peak height. A typical assay is shown in the tablebelow.

Allyl isothicyanate Peak Area Defatted Meal Sample 3 min 5 min 10 min 15min Brassica carinata 51 61 68 64 Brassica carinata + SG 91 264 282 284Sinapis alba + SG 189 347 291 273

The results indicate that exogenous sinigrin added to the defattedSinapis alba or Brassica carinata meal samples is rapidly converted toallyl-isothiocyanate under the conditions of the assay, confirming thepresence of active myrosinase in the processed samples. It should benoted that the conditions used to produce the defatted meal in thisexample were optimized for maximal oil recovery. Subsequent experimentscarried out to optimize conditions for obtaining maximal myrosinaseactivity have determined that heating of freshly ground meal samples at50° C. rather than 80° C. results in better preservation of myrosinaseactivity (data not shown).

Example 3 Assay for Glucosinolate Reduction from High Glucosinolate MealSource

A high glucosinolate lot of Brassica carinata meal (assayed to contain100.12 μmol/g total glucosinolate as determined using the InternationalOrganization for Standardization method reference number ISO 9167-3:2007 (E) Rapeseed—Determination of glucosinolate content—Part 3:Spectrometric method for total glucosinolates by glucose release) wasused to test the ability of the enzyme trigger solution to reduce theglucosinolate level. Samples of this meal were adjusted to variousmoisture contents by adding the solution of Sinapis alba meal (5% inwater containing buffer and ascorbic acid). The triggered meal sampleswere incubated at 55° C. for 30 min and extracted for HPLC analysis ofthe sinigrin content. As can be seen in the table below, the resultsshow that greater than 80% glucosinolate reduction can be obtained usingmoisture content and trigger meal concentration that is within theanticipated working range for processing.

% SG Sample SG Peak Area Reduction 20% Moisture 1735 55 25% Moisture 89877 30% Moisture 586 85 Control (untriggered) 3894 —

Example 4 Glucosinolate Reduction Carried Out at Room Temperature

The experiment described in Example 3 was repeated at room temperatureusing 30% moisture adjustment and compared with the performance of atrigger solution using the Brassica carinata meal prepared previously(BC/May) or the Sinapis alba meal prepared previously (SA/May). Theresults, summarized in the table below indicated that the batch ofdefatted Brassica carinata meal, prepared as described in Example 2 foruse as a trigger agent, had significant glucosinolate reductionactivity. The % SG Reduction is the reduction at 60 min.

SG Peak Area SG Peak Area % SG Sample (30 min, RT) (60 min, RT)Reduction 30% Moisture 949 700 82 SA/May 30% Moisture 1701 1552 60BC/May Control 3894 — — (untriggered)

Example 5 Glucosinolate Reduction Carried Out in the Absence ofBuffering Agents

Since the meal produced using this process is destined for animal feedit was important to minimize the addition of extraneous chemicalcompounds to the processing components (i.e. triggering solution).Therefore the activity of the trigger solutions with and without theaddition of buffers was assessed. The experiments were carried out using40% moisture and 60° C. incubation temperature. The results shown in thetable below indicate that the activity of the trigger solution whenapplied to dried meal at a level corresponding to 40% moisture is notstrongly dependent on the presence of buffers. The buffer used was 0.1 Mpotassium phosphate, pH 6.5. The % SG Reduction is the reduction at 60min.

SG Peak Area SG Peak Area % SG Sample (30 min, RT) (60 min, RT)Reduction 40% Moisture n/a 136 96.5 SA/May - Buffer + AA 40% Moisture182 115 97 SA/May - AA 40% Moisture n/a 328 91.6 BC/May - Buffer + AA40% Moisture 410 410 89.5 BC/May - AA Control 3894  — — (untriggered)

Example 6 Glucosinolate Reduction Under Desolventizing and ToastingConditions

Since the temperature of the meal in the stage of a typical crushingoperation may vary from input press cake temperature of ˜50° C. todesolventizing-toasting (DT) operating temperature of 90° C., theactivity of the trigger process was assessed using a temperaturegradient that would approximate conditions in the DT. An adjusted mealmoisture content of 35% was used and the temperature gradient shown inthe table below was applied. The results shown below indicate that thetrigger solution can theoretically yield glucosinolate reduction of themeal of greater than 75% when applied through the spray nozzle in thedesolventizing-toasting equipment that is present in most commercialseed crushing operations.

Temperature (° C.) Time (min) 55 2 65 3 75 3 85 12

Sample SG Peak Area % GLUCs Reduction SA/May 948 76 BC/May 1528 61Control (untriggered) 3894 —

Example 7 Pretreatment of Meal Prior to Glucosinolate Reduction

The assayed value of glucosinolate in large scale was obtained from abatch that was initially in a pellet form Hammer milling was carried outon the carinata meal samples to produce a uniform particle size prior tobatch processing in a Littleford™ dryer. The hammer milling was carriedout in a mill equipped with a 8/64″ screen (Model G5HFS1, serial number5075, Prater Industries, Chicago Ill.) Hammer-milled carinata meal wasdried to <12% moisture and then packaged into bulk sacks prior toglucosinolate removal processing.

Example 8 Batchwise Processing of Meal for Glucosinolate Reduction

13 batches of hammer-milled carinata meal samples were individuallyprocessed to remove glucosinolates as described below. The batchcharacteristics are as described in the tables below. For each batchprocess approximately 200 kg of meal was loaded into the Littleford™vacuum dryer (Littleford Reactor™ 600 liter model FKM600-D 2Z, serialnumber 5132, Littleford-Day Inc., Florence, Ky.). FIG. 2 is a schematicdiagram of the apparatus used for the batchwise removal of glucosinolatefrom Brassica carinata meal. The large dash box (a) indicates theLittleford vacuum dryer apparatus, which includes the reaction vessel 2for the batchwise treatment of meal for removal of glucosinolates. Theplough mixer was started and the meal heated to 35° C. 120 kg of softwater was loaded into a 200 liter stainless steel mixing vessel 6 and 45g of ascorbic acid was added. The dashed box in the lower right handcorner (b) indicates the mixing vessel 4 where the triggering solutionis prepared. After 5 minutes of mixing, 5 kg of defatted Sinapis albameal (batches 1-12) or Brassica carinata meal (batch 13) was added tothe mixing vessel 4 to form the triggering solution. The triggeringsolution was circulated via the inline mixer, comprising piping 20attached to a recirculation pump 22 downstream of drain 24, or 20minutes and then sprayed into the Littleford™ dryer reaction vessel 2where it was allowed to react with the carinata meal for 3 hours. Inbatch one, after the three hour reaction, a vacuum 46 was applied to thereaction vessel 2 and the meal was heated to 50° C. for one hour. Afterthe one hour hold at 50° C., the meal was heated to 70° C. -74° C. anddried to a moisture content of <12%. During batch one processing it wasnoted that the reaction was highly exothermic, so it was decided toremove the 1 hour hold at 50° C. and, instead, cooling water wascirculated in the dryer jacket (not visible) from the outset of thereaction to allow attainment of a maximum temperature at 50° C. -60° C.during the three hour reaction. From batch five onwards this was furthermodified so that cooling water was applied only once the temperature inthe reaction vessel 2 had reached 35° C. during the initial reaction.Also from batch 3 onwards, vacuum was applied from the onset of theinitial reaction. Once the processing for all batches was completed,small samples were removed for sinigrin peak height assay, while theremainder was packaged for storage. Samples of meal taken from theindividual processed batches were individually extracted and analyzed byHPLC as described in Example 1. The data of this analysis is summarizedin the table below.

SG Peak Area SG Peak Area of % SG Batch # Starting Material* TreatedFinal Product Reduction 1 4247 mean 482 88.65 2 4247 mean 489 88.49(2735 actual) (82.12) 3 4247 mean 482 88.65 4 4247 mean 496 88.32 5 4247mean 507 88.06 6 4247 mean 490 88.46 7 4247 mean 462 89.12 8 4247 Avg489 88.49 (5027 actual) (90.27) 9 4247 mean 499 88.25 10 4247 Avg 49688.32 (4979 actual) (90.04) 11 4247 mean 504 88.13 12 4247 mean 51287.94 13 (BC)** 4247 mean 501 88.20 *Starting material samples were onlyprovided for batches 2, 8, and 10. **Meal used in trigger solution wasBrassica carinata produced in the AGS lab

As can be seen, all processed batches gave greater than 80% reductionsin levels of glucosinolates relative to the starting material. Similarreductions were observed in batches 1 through 12, indicating that theheat generated by the exothermic reaction is sufficient to drive theprocess and that cooling only needed to be applied to keep the reactiontemperature from exceeding the 50° C.-60° C. ceiling. As well, it wasalso apparent that Brassica carinata based triggering solution wasequivalent to Sinapis alba based triggering solution in terms of itsability to catalyze the conversion of the glucosinolates.

Example 9 HPLC Analysis of Total GLS Content Samples of Carinata Meal

Quantitative HPLC analysis of meal samples were carried out on mealsamples prepared from commercial carinata variety A110 essentially asdescribed in Berhow et al., 2013. The meal samples consisted of: sample1: defatted meal prepared from A110 Brassica carinata seed as describedin example 2; samples 2,3: meal from a commercial crush of A110 Brassicacarinata seed processed via hexane extraction as described in thebackground section; samples 4,5: meal from a commercial crush of A110seed and further processed as described in Example 8.

uM/g uM/g uM/g uM/g uM/g SINIG SINA GNAP GNAS Sample ID GTRO DF DF DF DFDF 1 A110 Carinata A 0.00 68.72 0.00 5.15 0.38 (defatted meal) 2 A_1AP_APelleted 0.00 13.13 0.00 0.00 0.00 3 A_3BP_A Pelleted 0.00 18.75 0.914.19 0.00 4 P_B6/7_A 0.00 0.00 0.00 0.00 0.00 5 P_B8/9_A 0.00 0.00 0.000.00 0.00

In the above table, GTRO is progoitrin, Sinig is Sinigrin, Sina isSinalbin, GNAP is gluconapin, and GNAS is gluconasturtin. Thesemolecules are the different glucosinolates present in the meal samples.

As can be seen in the above table, meal prepared from seed crushed anddefatted in a lab scale process described in Example 2 (sample 1)performed with minimal heating during the procedure containedappreciable levels of glucosinolates consisting mainly of sinigrin (withlesser amounts of gluconapin and gluconasturtin). Meal of the same seedvariety but prepared from a commercial seed crush (samples 2, 3) wherethe cooking and flaking were carried out at elevated temperatures(greater than 105° C.) showed significant (68%-82%) reduction inglucosinolate levels relative to sample 1, whereas glucosinolate levelsof meal of the same seed variety obtained from a commercial crush andfurther processed using the procedure described in Example 8 (samples 4,5) were further reduced such that any remaining glucosinolate was belowthe limit of detection of the assay.

Example 10 Cooking Flaked Carinata Oilseed

In a research scale crush carried out on carinata A110 seed, a cookingstep was employed on the flaked seeds which comprised heating at 70° C.to 93° C.

Cooking is a standard practice carried out on the flaked seed wherebythe flakes are passed through stack-type cookers to disrupt oil cellsthat have survived flaking, reduce oil viscosity, denature hydrolyticenzymes and myrosinase, and adjust the moisture of the flakes prior topressing. Temperatures for cooking typically range from 80-105° C.

For the crush, the temperature was rapidly increased to 93° C. and thecycle was maintained for 15-20 minutes. The flaked carinata seeds thenunderwent crushing using an expeller, followed by hexane extraction torecover residual oil, followed by desolventing and toasting steps aspreviously described. The carinata meal produced by this process wasfound to have glucosinolate levels in the range of 110-120 μmol/g.

Example 11 Cooking Flaked Carinata Oilseed at Elevated Temperatures

In a subsequent crush carried out by a commercial crusher, cooking wascarried out on flaked carinata seed as described above, except that thetemperature of the cooking step exceeded 110° C., 115° C. or 120° C. Atthese temperatures, significant thermal degradation of glucosinolatesoccur, and indeed the meal produced by this crush was found to containglucosinolate levels of 13-20 μmol/g, significantly less than what wasobtained with the lower cooking temperature of the processing.

Meal obtained through a commercial crush as described in the previousparagraph was subsequently processed by batchwise treatment with anexogenous source of myrosinase (see Examples 7 and 8). The resultantmeal was shown to contain levels of glucosinolates that were below thelevels of detection of the HPLC based assay (see table in Example 9).

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1. A process for removing at least one glucosinolate from a mealfraction of oilseed comprising: (a) treating the meal fraction ofoilseed with exogenous myrosinase to convert the at least oneglucosinolate to a volatile isothiocyanate; and (b) removing thevolatile isothiocyanate from the treated meal fraction of oilseed underconditions of mild heat and negative pressure.
 2. The process of claim1, wherein step (a) and step (b) occur simultaneously or sequentially.3. The process of claim 1, wherein the time for step (a) and step (b) isgreater than 2 min, 5 min, 10 min, 15 min, 20 min, 30 min, 1 h, 2 h, 3h, or 4 h, and less than 9 h, 10 h, 11 h, or 12 h.
 4. The process ofclaim 1, wherein the process further comprises regulating thetemperature during step (a) and step (b) to prevent the temperature fromexceeding 45° C., 50° C., 55° C., 60° C., 65° C., or 70° C.
 5. Theprocess of claim 1, wherein the process further comprises carrying outstep (a) and step (b) in a reaction vessel under negative pressure ofover 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%vacuum and evacuating volatile substances from the reaction vessel. 6.The process of claim 1, wherein the process further comprises increasingthe temperature to over 60° C., 65° C., 70° C., 75° C., 80° C., 85° C.,or 90° C., but to less than 95° C., 100° C., 105° C., 110° C., or 115°C., following step (a) and step (b) and further incubating the mealfraction of oilseed until the moisture content of the meal fraction ofoilseed decreases to less than 20%, 18%, 16%, 14%, 12%, or 10%.
 7. Theprocess of claim 6, wherein the process further comprises carrying outthe further incubation in a reaction vessel under negative pressure ofover 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%vacuum and evacuating volatile substances from the reaction vessel. 8.The process of claim 1, wherein the process further comprisescontinuously mixing the meal fraction of oilseed and exogenousmyrosinase.
 9. The process of claim 1, wherein the process furthercomprises preheating the meal fraction of oilseed to between 25° C. to40° C. prior to step (a).
 10. The process of claim 1, wherein theprocess further comprises providing the exogenous myrosinase in the formof a triggering solution comprising a slurry of defatted meal in water.11. The process of claim 10, wherein the defatted meal is from oilseedof a plant species of the Brassicaceae family.
 12. The process of claim11, wherein the defatted meal is from oilseed of Sinapis alba orBrassica carinata.
 13. The process of claim 10, wherein the triggeringsolution further comprises ascorbic acid.
 14. The process of claim 10,wherein the process further comprises adding triggering solution to themeal fraction of oilseed in a ratio of 0.5:1, 0.55:1, 0.6:1, 0.65:1, or0.7:1 (w/w) triggering solution: meal fraction of oilseed.
 15. Theprocess of claim 1, wherein the meal fraction of oilseed results fromhexane extraction of oilseeds.
 16. The process of claim 15, wherein thehexane extraction was carried out on flaked and cooked oilseed, andwherein the cooking was carried out at a temperature greater than 70°C., 75° C., 80° C., 85° C., 90° C., 95° C., 100° C., 105° C., 110° C.,115° C., 120° C., 125° C., 130° C., 135° C., or 140° C., but less than150° C., 155° C., 160° C., 165° C., 170° C., 175° C., or 180° C.
 17. Theprocess of claim 15, wherein the hexane extraction was carried out onflaked and cooked oilseed, and wherein the duration of cooking was atleast 10, 12, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, or 30 minutes,but less than 60, 70, 80, 90, or 100 minutes.
 18. The process of claim15, wherein the hexane extraction was carried out on flaked and cookedoilseed that had been pressed using an expeller or a screw press. 19.The process of claim 15, wherein the hexane extraction was carried outon flaked and cooked oilseed, and wherein the resultant meal cakeunderwent treatment in a desolventizer-toaster at a temperature of atleast 50° C., 55° C., 60° C., 65° C., 70° C., 75° C., 80° C., 85° C.,90° C., 95° C., 100° C., 105° C., 110° C., 115° C., 120° C., 125° C., or130° C., but less than 135° C., 140° C., 145° C., or 150° C.
 20. Theprocess of claim 15, wherein the reaction vessel is adesolventizer-toaster, and wherein step (a), step (b), and the furtherincubation are part of a desolventizer-toaster step of hexane extractionof oilseeds.
 21. The process of claim 1, wherein the meal fraction ofoilseed results from cold press processing of oilseeds.
 22. The processof claim 1, wherein the meal fraction of oilseed to be treated ispelleted and homogenized by hammer-milling to a size of less than 5.66mm, 4.75 mm, 4.00 mm, 3.36 mm, 2.83 mm or 2.38 mm.
 23. The process ofclaim 1, wherein the meal fraction of oilseed is from oilseed of a plantspecies of the family Brassicaceae.
 24. The process of claim 1, whereinthe meal fraction of oilseed is from oilseed of Brassica juncea,Brassica napus, Brassica rapa, Brassica carinata, Brassica nigra,Cammelina sativa, Crambe abyssinica, or Thlaspi arvense.
 25. A processfor removing at least one glucosinolate from a meal fraction of aBrassica carinata oilseed comprising heating and applying pressure tothe oilseed before, during, or after the extraction of oil.
 26. Theprocess of claim 25, wherein the temperature during heating is greaterthan 70° C., 75° C., 80° C., 85° C., 90° C., 95° C., 100° C., 105° C.,110° C., 115° C., 120° C., 125° C., 130° C., 135° C., or 140° C., butless than 150° C., 155° C., 160° C., 165° C., 170° C., 175° C., or 180°C.
 27. The process of claim 25, wherein the duration of heating is atleast 10, 12, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, or 30 minutes,but less than 60, 70, 80, 90, or 100 minutes.
 28. The process of any oneof claim 25, wherein the process further comprises applying the pressureusing an expeller or a screw press.
 29. A process for removing at leastone glucosinolate from a meal fraction of a Brassica carinata oilseedcomprising: (a) heating and applying pressure to the oilseed before,during, or after the extraction of oil; (b) treating the meal fractionof oilseed with exogenous myrosinase to convert the at least oneglucosinolate to a volatile isothiocyanate; and (c) removing thevolatile isothiocyanate from the treated meal fraction of oilseed underconditions of mild heat and negative pressure.
 30. The process of claim29, wherein the temperature of heating in step (a) is greater than 70°C., 75° C., 80° C., 85° C., 90° C., 95° C., 100° C., 105° C., 110° C.,115° C., 120° C., 125° C., 130° C., 135° C., or 140° C., but less than150° C., 155° C., 160° C., 165° C., 170° C., 175° C., or 180° C.
 31. Theprocess of claim 29, wherein the duration of heating in step (a) is atleast 10, 12, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, or 30 minutes,but less than 60, 70, 80, 90, or 100 minutes.
 32. The process of claim29, wherein the process further comprises applying the pressure in step(a) using an expeller or a screw press.